Alcohol dehydrogenase variants

ABSTRACT

Described herein are non-natural NAD+-dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. Also described are engineered cells expressing a non-natural NAD+-dependent alcohol dehydrogenase, optionally include one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, target product pathway genes, cell culture compositions including the cells, methods for promoting production of the target product or intermediate thereof from the cells, compositions including the target product or intermediate, and products made from the target product or intermediate.

PRIORITY CLAIM

This application claims the benefit of International Application No.PCT/US2014/059135, filed Oct. 3, 2014, which in turn claims the benefitof U.S. Provisional Patent Application Ser. No. 61/887,251 filed Oct. 4,2013, entitled ALCOHOL DEHYDROGENASE VARIANTS, the entireties of saidpatent applications are incorporated herein by reference. Also, theentire contents of the ASCII text file entitled“GNO0003WO_Sequence_Listing_V2_ST25.txt” created on Aug. 26, 2019,having a size of 207 kilobytes is incorporated herein by reference.

BACKGROUND

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) promote the conversion ofalcohols to and aldehydes or ketones, typically along with the reductionof nicotinamide adenine dinucleotide (NAD⁺ to NADH). ADHs areinstrumental in the generation of important compounds having aldehyde,ketone, and alcohol groups during biosynthesis of various metabolites.

One class of alcohol dehydrogenase is methanol dehydrogenases (MDHs).MDHs, converts methanol (MeOH) to formaldehyde (Fald), may be used in anenzymatic pathway engineered into a microbe to enable MeOH as a solecarbon source or as a co-carbon source with other feed stocks such as,for example, glucose, dextrose, plant biomass or syngas, to producevaluable products. Microorganisms have been reported that metabolizemethanol, and in some instances do so via a methanol dehydrogenase, andin even fewer instances produce valuable products. Increasing MDHactivity will enable improved use of MeOH, improving MeOH as a solecarbon source, decreasing production costs, decreasing amounts of anymore expensive secondary or co-carbon source, e.g. glucose, increasingproduct yields, and providing faster rate of MeOH use.

SUMMARY

Generally, presented herein are non-natural NAD⁺-dependent alcoholdehydrogenases (ADHs) (i.e. engineered enzymes and theirencoding-polynucleotides) capable of at least two fold greaterconversion of methanol or ethanol to formaldehyde or acetaldehyde,respectively, as compared to its original or unmodified counterpart.Exemplary aspects describe non-natural NAD⁺-dependent methanoldehydrogenases (MDHs), in particular enzymes of the class EC 1.1.1.244.

The ADHs and MDHs have at least one amino acid substitution as comparedto its corresponding natural or unmodified alcohol dehydrogenase. Byunmodified alcohol dehydrogenase is meant that the ADH or MDH may havebeen previously engineered (e.g., need not be naturally-occuring), priorto incorporating any modification described herein. Such alcoholdehydrogenases that are starting sequences for incorporating amodification described herein to generate the novel engineered enzymemay be alternatively referred to herein as wild-type, template, startingsequence, natural, naturally-occurring, unmodified, correspondingnatural alcohol dehydrogenase, corresponding natural alcoholdehydrogenase without the amino acid substitution, corresponding alcoholdehydrogenase or corresponding alcohol dehydrogenase without the aminoacid substitution. Experimental studies described herein demonstrate forthe first time that a number of amino acid positions along the length ofthe amino acid sequence can be substituted to provide a non-naturaldehydrogenase having increased substrate conversion. The studies alsoshow that combinations of substitutions (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve, etc.) in an amino acidsequence can also provide even further increased substrate conversion.Provided herein therefore are single and combination variants of astarting or template or corresponding alcohol dehydrogenase, e.g., inparticular enzymes of the class EC 1.1.1.244, having increased substrateconversion.

Embodiments of the disclosure provide a non-natural NAD⁺-dependentalcohol dehydrogenase comprising at least one amino acid substitution ascompared to a corresponding alcohol dehydrogenase, and capable of atleast two fold greater conversion of methanol or ethanol to formaldehydeor acetaldehyde, as measured relative to a corresponding alcoholdehydrogenase without amino acid substitution. Embodiments of thedisclosure also provide a non-natural NAD⁺-dependent alcoholdehydrogenase comprising at least one amino acid substitution capable ofat least two fold greater conversion of methanol or ethanol toformaldehyde or acetaldehyde, respectively, as compared a secondsequence that is an NAD⁺-dependent alcohol dehydrogenase, wherein thefirst and second sequences differ with regards to the at least one aminoacid substitution. Embodiments are also directed to engineered cellsexpressing the non-natural NAD⁺-dependent alcohol dehydrogenase capableof at least two fold greater conversion of methanol or ethanol toformaldehyde or acetaldehyde as described.

Some embodiments of the current disclosure are directed to an engineeredcell expressing a non-natural NAD⁺-dependent alcohol dehydrogenasecomprising at least one amino acid substitution (including single andcombination variants). The cells can be used to promote production of atarget product or intermediate thereof. For example, the cell mayprovide either or both an increased amount of reducing equivalents, e.g.NADH, for an increase in a target product or may provide for increasedfixation of carbon from formaldehyde and/or acetaldehyde into a targetproduct. Exemplary products include (a) 1,4-butanediol and intermediatesthereto, such as 4-hydroxybutanoic acid (4-hydroxybutanoate,4-hydroxybutyrate, 4-HB), (b) butadiene and intermediates thereto, suchas 1,4-butanediol, 1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methylvinyl carbinol) and 3-buten-1-ol, (c) 1,3-butanediol and intermediatesthereto, such as 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol, (d)adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine andlevulinic acid and their intermediates, e.g. 4-aminobutyryl-CoA, (e)methacrylic acid (2-methyl-2-propenoic acid) and its esters knowncollectively as methacrylates, such as methyl methacrylate, methylmethacrylate, 3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and theirintermediates, (f) 1,2-propanediol (propylene glycol), n-propanol,1,3-propanediol and glycerol, and their intermediates and (g) succinicacid and intermediates thereto.

Embodiments of the engineered cell may further optionally include one ormore additional metabolic pathway transgene(s) to further promoteproduction of the target product or intermediate thereof. In exemplaryembodiments the cell further comprises one or more methanol metabolicpathway (MMP) transgene(s), such as a formaldehyde dehydrogenasetransgene, allowing expression of the encoded pathway enzyme oraccessory protein.

In exemplary embodiments the cell further comprises a product pathwaycomprising enzymes (and their endoding polynucleotides) for productionof a target product, such as the enzymes described herein for productionof 1,4-butanediol from glucose.

Other embodiments are directed to compositions including engineeredcell, such as cell culture compositions, and also compositions includingone or more product(s) produced from the engineered cell. For example, acomposition can include a target product or intermediate thereofproduced by the cells, where the composition has been purified to removecells or other components useful for cell culturing. The composition maybe treated to enrich or purify the target product or intermediatethereof.

Other embodiments of the disclosure are directed to products made fromthe target product obtained from methods using the engineered cell.Exemplary products include polymers made with target products, such aspolymers made from diol target products combined with diacids, includingtarget product succinic acid, such as polybutylene terephthalate (PBT)and polybutylene succinate (PBS) made from 1,4-butanediol polymerizedwith terephthalic acid or succinic acid respectively.

Other embodiments of the disclosure are directed to nucleic acidsencoding the non-natural alcohol dehydrogenases with one or more variantamino acids, as well as expression constructs including the nucleicacids, and engineered cells comprising the nucleic acids or expressionconstructs.

In other embodiments the disclosure also provides methods for generatingnon-natural NAD⁺-dependent alcohol dehydrogenases capable of at leasttwo-fold greater conversion of methanol or ethanol to formaldehyde oracetaldehyde, respectively, as compared to its unmodified (original;template) counterpart. In some embodiments, the method includes steps of(a) identifying a variant amino acid that provides increased conversionin a template sequence, (b) identifying corresponding amino acidposition in a target sequence having identify to the template sequence,and (c) changing the amino acid at the corresponding amino acid positionin a target sequence to the variant amino acid. The starting templatefor incorporation of modifications described herein can be anaturally-occurring enzyme sequence or a previously engineered enzymesequence.

In other embodiments, the methods includes steps of (a) identifying anamino acid position in a non-natural NAD⁺-dependent alcoholdehydrogenases that is not a variant position, (b) providing, in aoriginal template, a variation at an amino acid position that is anon-variant position, and (c) identifying variants from step (b) thatprovide increased conversion of the substrate.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are graphs listing amino acid positions of methanoldehydrogenase (2315A) and the effect of substitution of those positionson enzyme activity.

FIG. 2 illustrates a pathway using MDH to produce 1, 4-butanediol (BDO)in organism such as E. coli.

FIGS. 3A-D illustrate pathways using certain methanol metabolizingenzymes. FIG. 3D illustrates using enzymes to fix carbon from methanolvia formaldehyde assimilation into a product pathway of interest. The“Product Pathway” can be that of 1,4-butanediol as described herein orother product pathway.

FIG. 4 is an amino acid sequence alignment of various Fe-dependentalcohol dehydrogenases with Bacillus MeDH.

DETAILED DESCRIPTION

The embodiments of the description described herein are not intended tobe exhaustive or to limit the disclosure to the precise forms disclosedin the following detailed description. Rather, the embodiments arechosen and described so that others skilled in the art can appreciateand understand the principles and practices of the description.

All publications and patents mentioned herein are hereby incorporated byreference. The publications and patents disclosed herein are providedsolely for their disclosure. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate anypublication and/or patent, including any publication and/or patent citedherein.

Generally, the disclosure provides non-natural NAD⁺-dependent alcoholdehydrogenases (ADHs) capable of at least two fold greater conversion ofmethanol or ethanol to formaldehyde or acetaldehyde, respectively, ascompared to its unmodified counterpart. Nucleic acids encoding thenon-natural alcohol dehydrogenases, as well as expression constructsincluding the nucleic acids, and engineered cells comprising the nucleicacids or expression constructs are described.

Also described are engineered cells expressing a non-naturalNAD⁺-dependent alcohol dehydrogenase, optionally including one or moreadditional metabolic pathway transgene(s), methanol metabolic pathwaygenes, and/or target product pathway genes; cell culture compositionsincluding the cells; methods for promoting production of the targetproduct or intermediate thereof from the cells; compositions includingthe target product or intermediate; and products made from the targetproduct or intermediate.

The term “non-naturally occurring”, when used in reference to anorganism (e.g., microbial) is intended to mean that the organism has atleast one genetic alteration not normally found in a naturally occurringorganism of the referenced species. Naturally-occurring organisms can bereferred to as “wild-type” such as wild type strains of the referencedspecies. Likewise, a “non-natural” polypeptide or nucleic acid caninclude at least one genetic alteration not normally found in anaturally-occurring polypeptide or nucleic acid. Naturally-occurringorganisms, nucleic acids, and polypeptides can be referred to as“wild-type” or “original” such as wild type strains of the referencedspecies. Likewise, amino acids found in the wild type organism can bereferred to as “original” with regards to any amino acid position.

A genetic alteration that makes an organism non-natural can include, forexample, modifications introducing expressible nucleic acids encodingmetabolic polypeptides, other nucleic acid additions, nucleic aciddeletions and/or other functional disruption of the organism's geneticmaterial. Such modifications include, for example, coding regions andfunctional fragments thereof, for heterologous, homologous or bothheterologous and homologous polypeptides for the referenced species.Additional modifications include, for example, non-coding regulatoryregions in which the modifications alter expression of a gene or operon.

An NAD(P)+-dependent methanol dehydrogenase from Bacillus methanolicusMGA3 (Genbank Accession number EIJ77596.1, GI number: 387585261;designated herein as MDH 2315, 382 amino acids long; SEQ ID NO: 1), wasselected as a template for mutagenesis to identify variants withimproved activity. This sequence was selected as it was surprisinglyfound to be very active on methanol, typically nearly twice as active asother alcohol dehydrogenases tested, and used NAD+ and thus able toregenerate NADH that can be useful to enzymes in target pathways. MDH2315 is reported in the literature as an NAD(P)-dependent methanoldehydrogenase from Bacillus methanolicus MGA3 and its sequence wasdescribed in Brautaset et al., “Plasmid-Dependent Methylotrophy inThermotolerant Bacillus methnolicus”, Journal of Bacteriology, vol. 186,pp 1229-1238 (2004). It is also referred to as MDH MGA3 in WO2013/110797to Brautaset and MDH “M” in Krog et al., “Methylotrophic Bacillusmethanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ DependentMathanol Dehydrogenase Paralogs with Different Catalytic and BiochemicalProperties”, PLOS ONE, pp. 1-11, (2013), which report addititionalwild-type Bacillus MDHs.

MDH 2315A was expressed in E. coli and a library of variants wasgenerated by error prone PCR (the epPCR Library). Specifically, MDH2315A was expressed in E. coli and subjected to saturation mutagenesisat 375 of 382 positions to generate a library of all single pointsubstitutions of the common 20 amino acids (the NNK library).

In the primary screen, the NNK Library was screened by assayingindividual colony cell extracts for MeOH to Fald conversion. The librarywas screened for variants that had greater than 2-fold activity overwild-type (defined as a positive). Such a library can contain variantswith multiple mutations, deletions, etc. However, variants with singlepoint substitutions were identified. In that primary screen about 150positions gave colonies whose extracts had activity reliably 2-foldgreater than wild-type. Of those, 21 positions gave colonies withactivity reliably greater than 4-fold wild-type.

The primary screen of the NNK library identified about 10 positionswhere 90% or more of the colonies were inactive, suggesting that anyamino acid other than wild-type at those positions led to enzymeinactivity (or rapid degradation). About 30 positions were identified inwhich less than 5% of the colonies were inactive. Many positions wereidentified in which no positives were observed but the total number ofinactives was less than 90%. Many positions were identified in which thetotal number of positives and inactives were less than 95%.

Secondary in vitro assays in triplicate and sequencing were done on allpositives greater than 4-fold, and many greater than 2-fold activity. At40 positions, a total of 90 substitutions were determined to beresponsible for the activity increase either greater than 2-fold or inmany cases greater than 4-fold, and up to 10-fold. In many positions,only a single specific amino acid gave improvement, in other positionsseveral amino acid substitutions gave improvement (e.g., at 213 onlyN213D, but at 121 both G121D, G121V, etc).

Secondary screening of the epPCR library revealed about 20 positionswhose substitutions gave activity greater than 3-fold wild-type.Sequencing of these colonies revealed the specific substitutions at eachposition, for example, N213D (nomenclature: N is original or unmodifiedamino acid at position 213; D is the substitution). At these 20positions, a total of 30 substitutions provided greater than 3-foldwild-type activity of which about 15 variants provided greater than4-fold activity.

Some of the colonies whose MDH variant sequence were identified werealso assayed in vivo by measuring MeOH conversion to formaldehyde in astrain background in which genes for use of formaldehyde wereinactivated, thus enabling accumulation of formaldehyde. Generally, allMDH variants that were positive (greater than wild-type) in in vitroscreening also enabled greater than wild-type activity in vivo, althoughthe in vitro to in vivo activities did not always correlate exactly.

At some positions the frequency of a particular substitution in thetotal population of colonies at that position in a library was zero orless than statistically expected. Thus some substitutions at somepositions may not have been present or present and not detected.

Table 1 lists amino acids mutations with respect to SEQ ID NO: 1,providing greater than two fold activity when present as singlemutations. While not to be bound by theory, as depicted in FIG. 1,functional features associated with amino acid positions of methanoldehydrogenase designated 2315A and corresponding positions in othermethanol dehydrogenase described herein include: NADH cofactor bindingcorrelating with positions 38D, 39A, 40F, 70D, 97G, 98S, 137T, 138T,141T, 142G, 143S, 145T, 146T, 147S, 148L, 149A, 150V, 161P, 162V, 163I;activation site correlating with positions 95G, 97G, 98S; and substratecorrelating with positions 145T, 146T, 147S, 148L, 149A, 150V, 161P,162V, 163I, 253F, 258L, 266H, 359D, 360V, 361C.

As can be seen from FIG. 1 depicting site saturation mutagenesis,numerous amino acid positions of methanol dehydrogenase designated 2315Aare tolerant to substitution (indicated by a percentage of colonieshaving greater than 2-fold (designated “hits”) or from 0.2 to less than2-fold activity of wild-type enzyme) and others less tolerant (indicatedby percentage of colonies where enzyme activity was less than 20% ofwild-type enzyme activity (designated “dead”). For example, 168T and270G are very tolerant to change, where substitutions at 168T generallyhad little effect on activity, whereas substitutions at 270Gpredominantly improved activity.

Art known methods can be used for the testing the enzymatic activity ofalcohol dehydrogenases, and such methods can be used to test activity ofalcohol dehydrogenase (ADH) variant enzymes as well. As a generalmatter, a reaction composition including the alcohol dehydrogenase (ADH)variant, an alcohol (substrate) and NAD (cofactor) can be converted to adehydrogenated product. For example, conversion of ethanol is shown asfollows:

Reaction can be carried out at a desired temperature, such as 25° C.,and pH, such as pH 7.

The ADH variant, can be defined in terms of its enzymatic activity withone unit of enzyme converting 1.0 μmole of alcohol to dehydrogenatedproduct per minute at pH 8.8 @ 25° C. See, for example, Kagi, J. H. R.and Vallee, B. L. (1960) Journal of Biological Chemistry 235, 3188-3192

Of particular interest herein is conversion of methanol to formaldehydeto regenerate NADH. This conversion can be followed by either or bothconversion to formate or fixation of the formaldehyde carbon into targetproduct. The formate can be either or both converted to CO₂ or have itscarbon fixed into target product, such as by conversion back toformaldehyde. See the attached figures.

A representative in vivo assay was developed to determine the activityof methanol dehydrogenase variants in organisms is reported in U.S.application Ser. No. 13/975,678. This assay relies on the detection offormaldehyde (Palp), thus measuring the forward activity of the enzyme(oxidation of methanol). To this end, a strain comprising a BDOP andlacking frmA, frmB, frmR was created using Lamba Red recombinasetechnology (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA, 6 97(12):6640-5 (2000). Plasmids expressing methanol dehydrogenases weretransformed into the strain, then grown to saturation in LBmedium+antibiotic at 37° C. with shaking. Transformation of the strainwith an empty vector served as a negative control. Cultures wereadjusted by O.D. and then diluted 1:10 into M9 medium+0.5%glucose+antibiotic and cultured at 37° C. with shaking for 6-8 hoursuntil late log phase. Methanol was added to 2% v/v and the cultures werefurther incubated for 30 min. with shaking at 37° C. Cultures were spundown and the supernatant was assayed for formaldehyde produced usingDETECTX Formaldehyde Detection kit (Arbor Assays; Ann Arbor, Mich.)according to manufacturer's instructions. The frmA, frmB, frmR deletionsresulted in the native formaldehyde utilization pathway to be deleted,which enables the formation of formaldehyde that can be used to detectmethanol dehydrogenase activity in the organism. These genes are deletedin this case solely to facilitate measurement of methanol conversion bypreventing loss of the measured analyte, formaldehyde.

Enzymatic kinetic assays were done for 10 single point variants, andthat 2-10 fold improvements in activity were reflected in 2-10 foldimprovements in Km, Vmax or both. Cofactor binding nor substrate orproduct on-off rates were not measured.

Table 7 shows enzymology data from various wild type ADH proteins.Tables 8 and 9 show data for wild type and variant enzymes, with Table 8showing activity using either methanol or 1,4-butanediol, and Table 9showing 1,4-butanediol-dependent steady-state kinetic parameters forwild-type and variant methanol dehydrogenase.

Results of the mutagenesis procedures and rationale design and screeningof the positives (by “positive” is meant a sequence modified asdescribed herein having at least a two (2) fold increase in activitycompared to the unmodified template sequence) revealed a number of aminoacid variants along the MDH protein 2315A template for use in theinvention. Positives showing greater than two fold increase in activityare shown in Table 1, and listed as follows: S11T, D38N, H42Q, E48D,N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G,K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W,V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R,G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W,G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L,S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R,P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T,T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W,A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E,V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N,I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S,I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S,R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D,V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A,V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M. Thesechanges, their positions in SEQ ID NO: 1, and their correspondingpositions in other template sequences are described further in thetables and elsewhere herein.

Of more interest are positives showing greater than two fold increase inactivity as single mutations shown in Table 1, and listed as follows:D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y,R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S,G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y,S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R,P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T,T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K,V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G,V360K, V360R, V360S, C361R. These changes, their positions in SEQ ID NO:1, and their corresponding positions in other template sequences aredescribed further in the tables and elsewhere herein.

Results of the rationale design mutagenesis procedures and the otherlibrary generation procedures described herein and screening of thepositive revealed a number of combination amino acid variants along theMDH protein 2315A template. Positives showing greater than two foldincrease in activity are shown in Tables 2-4, and listed as variationsin the following sets: (a) D70N, L148G, P161G, V360A; (b) D70N, L148G,V360A, C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V,V360G; (e) D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N,V150I, P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D,L148V, P161A, V360A; (j) L148G, P161A, V360A, C361N; (k) L148G, P161A,V360G; (l) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n)L148G, P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G,C361N; (q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T,V150I, V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V,V150I, P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V,V150I, P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V,V150I, P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V,V150I, P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K,S147R, P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A,C361N; (ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A,C361N; (aj) L148W, P161A, V360A, C361N; (ak) N112K, S147R, P161A, V360A;(al) P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A,V360G; (ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V,V360G; (ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A,V150I, P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R,L148V, P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A,V360G, C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F,V150I, P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R,L148V, P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac)S147R, L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae)S147R, P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R,P161V, V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I,P161V, V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I,V360G; (aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N;(aan) V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap)V150I, P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I,P161V, K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat)V150I, P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I,V360G; (aaw) S11T, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A,I163N; (aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F,N355K; (aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I,V158E; (aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N;(aaai) A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal)A155V, R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao)G121A, T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G;(aaar) Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau)L226M, A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G,V158E; (aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A,V158E; (aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V;(aaaad) K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag)S124R, E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S,F333L; and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V,(aaaan) D73D/L108V, (aaaao) G121R/P161S, and (aaaap) N112R/P161S.

Also identified were positions in SEQ ID NO:1 that are generallyintolerant to substitution, including P8, V132, E177, A207, T208, Q246,I264, P285, E308, Y339, A340, A353, T362, R367, P369, D373, and I377;where original amino acids are of particular interest at these positionsin SEQ ID NO:1 and their corresponding positions in other templates asdescribed herein.

Also identified were positions in SEQ ID NO:1 that are generally verytolerant to substitution, including E56, E78, E86, T2, N4, E237, E240,E327, E341, E347, P9, V18, R24, T44, L46, K52, D60, A62, F64, A67, P71,A72, D73, T74, V80, K84, Q85, I106, V109, R115, I116, N117, V122, K127,I135, T136, S154, A155, R156, P161, V162, I163, P169, T170, V171, V174,L178, M179, A184, G185, L186, A189, T190, A194, A198, Y202, V203, T211,F214, I216, Q217, K220, L221, N223, Y225, A229, Y244, A245, M248, A252,N254, H266, G270, Y272, I278, M284, H286, V287, N291, I293, A294, R298,H301, I302, L305, N309, A311, G312, T315, A317, R321, V324, I329, S332,S337, M342, K345, K354, N355, A356, Y357, T370, A375, and I378 in SEQ IDNO:1 and their corresponding positions in other templates; where it wasobserved that typically all amino acids, except for occasionally 1 or 2,were neutral, beneficial, or less than an 80% decrease to activity atthese positions. Of these, D60, I378, L46, A62, A67, N117, K52, I135,Y202, I216, M248, N291, M342, T2, E240, T44, I116, V203, S332, and thustheir corresponding positions in other templates, were tolerant to allchanges.

Positions for additional interest for substitution to increase activityinclude 23T, 81D, 141T, 174V, 189A, 332S, 372Q and 379K and theircorresponding positions in other templates.

Embodiments of the disclosure are also directed to the preparation anduse of other alcohol dehydrogenase (ADH) variant enzymes includingmethanol dehydrogenases variant enzymes, based on the single andcombination amino acid variants indentified in and with respect to theMDH protein 2315A template. In these embodiments generation ofnon-natural NAD⁺-dependent alcohol dehydrogenases (that are not 2315A)capable of at least two fold greater conversion of methanol or ethanolto formaldehyde or acetaldehyde, respectively, as compared to itsoriginal unmodified counterpart can be based on information of those MDH2315A variants that provided increased activity.

In some embodiments, the method for preparing other ADH variantsincludes steps of (a) identifying a variant amino acid that providesincreased conversion when present in the 2315A template sequence, (b)identifying a corresponding amino acid position in a target sequence(i.e., other ADH or MDH sequence) having identity to the templatesequence, and (c) changing the amino acid at the corresponding aminoacid position in the target sequence to the variant amino acid. Forexample, the D at position 38 of 2315A when substituted with N providesgreater than two fold increase in activity compared to the unmodified2315A sequence. A position corresponding to D38 is identified in the newtarget ADH or MDH template sequence, e.g., it may be D38 or D39appropriate, and replaced with N to generate the newnon-naturally-occurring ADH or MDH.

In some cases an “ortholog” of the NAD(P)-dependent methanoldehydrogenase from Bacillus methanolicus MGA3 (2315A), SEQ ID NO: 1, isfirst identified. An ortholog is a gene or genes that are related byvertical descent and are responsible for substantially the same oridentical functions in different organisms. Genes are related byvertical descent when, for example, they share sequence similarity ofsufficient amount to indicate they are homologous, or related byevolution from a common ancestor. Genes can also be considered orthologsif they share three-dimensional structure but not necessarily sequencesimilarity, of a sufficient amount to indicate that they have evolvedfrom a common ancestor to the extent that the primary sequencesimilarity is not identifiable. Genes that are orthologous can encodeproteins with sequence similarity of about 45% to 100% amino acidsequence identity, and more preferably about 60% to 100% amino acidsequence identity.

Genes sharing a desired amount of identify (e.g., 45%, 50%, 55%, or 60%or greater) to the NAD(P)-dependent methanol dehydrogenase from Bacillusmethanolicus MGA3 (2315A), including orthologs and paralogs, can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.

Computational approaches to sequence alignment and generation ofsequence identity include global alignments and local alignments. Globalalignment uses global optimization to forces alignment to span theentire length of all query sequences. Local alignments, by contrast,identify regions of similarity within long sequences that are oftenwidely divergent overall. For understanding the indentity of a targetsequence to the Bacillus methanolicus MGA3 (2315A) template a globalalignment can be used. Optionally, amino terminal and/orcarboxy-terminal sequences of the target sequence that share little orno identify with the template sequence can be excluded for a globalalignment and generation of an identify score.

Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 45% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).

Pairwise global sequence alignment was carried out for each of thetemplate polypeptides with SEQ ID No.1 (2315A) as the reference. Thealignment was performed using the Needleman-Wunsch algorithm (Needleman,S. & Wunsch, C. A general method applicable to the search forsimilarities in the amino acid sequence of two proteins J. Mol. Biol,1970, 48, 443-453) implemented through the BALIGN tool(balign.sourceforge.net). Default parameters were used for the alignmentand BLOSUM62 was used as the scoring matrix.

Table 10 provides target polypeptides details and alignment to SEQ IDNO. 1 (2315A). These sequences represent target sequences in which oneor more amino acid variations, based on the variant amino acids in theBacillus methanolicus MGA3 (2315A) variants showing increasedconversion, can be made. For example, as a general matter, this processcan involve steps of aligning the template 2315A sequence to a targetsequence, such as any sequence listed in Table 10. Next a position ofthe amino acid substitution/variant (or set of substitutions) in thetemplate 2315A sequence providing the increased conversion of methanolor ethanol is identified. The amino acid alignment at thesubstitution/variant position is inspected to identify what amino acidposition in the target sequence corresponds to that of the template2315A sequence. Preferred target sequences for substitution with theamino acid variants based on the 2315A variants are highlighted.

In some cases the original amino acid and its position on the template2315A sequence will precisely correlate with the original amino acid andposition on the target. In other cases the original amino acid and itsposition on the template 2315A sequence will correlate with the originalamino acid, but its position on the target will not be in thecorresponding template position. However, the corresponding amino acidon the target can be a predetermined distance from the position on thetemplate, such as within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidpositions from the template position. In other cases the original aminoacid on the template 2315A sequence will not precisely correlate withthe original amino acid on the target. However one can understand whatthe corresponding amino acid on the target sequence is based on thegeneral location of the amino acid on the template and the sequence ofamino acids in the vicinity of the target amino acid. For example, aminoacids in the vicinity of the target amino acids may be viewed as a“sequence motif” having a certain amount of identity or similarity tobetween the template and target sequences.

In some cases, it can be useful to use the Basic Local Alignment SearchTool (BLAST) algorithm to understand the sequence identity between anamino acid motif in a template sequence and a target sequence.Therefore, in preferred modes of practice, BLAST is used to identify orunderstand the identity of a shorter stretch of amino acids (e.g. asequence motif) between a template and a target protein. BLAST findssimilar sequences using a heuristic method that approximates theSmith-Waterman algorithm by locating short matches between the twosequences. The (BLAST) algorithm can identify library sequences thatresemble the query sequence above a certain threshold. Exemplaryparameters for determining relatedness of two or more sequences usingthe BLAST algorithm, for example, can be as set forth below. Briefly,amino acid sequence alignments can be performed using BLASTP version2.0.8 (Jan. 5, 1999) and the following parameters: Matrix: 0 BLOSUM62;gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize:3; filter: on. Nucleic acid sequence alignments can be performed usingBLASTN version 2.0.6 (Sep. 16, 1998) and the following parameters:Match: 1; mismatch: −2; gap open: 5; gap extension: 2; x_dropoff: 50;expect: 10.0; wordsize: 11; filter: off. Those skilled in the art willknow what modifications can be made to the above parameters to eitherincrease or decrease the stringency of the comparison, for example, anddetermine the relatedness of two or more sequences.

In view of and following the teachings herein, using methods known inthe art such as sequence alignment and 3D modeling, the “correspondingpositions and amino acids” for substitution in template polypeptidesother than SEQ ID NO: 1 are readily determined. Table 11 indicates foreach template the corresponding positions for substitution to improvepolypeptide activity, from which each original amino acid, its locationand substitution is specifically contemplated as if expressly listed.For example, using the 385 amino acid template polypeptide ofEIJ83020.1, GI:387590701 (SEQ ID NO:7), from Bacillus methanolicus MGA3,having 61% global identity and 79% similarity to SEQ ID NO:1, theformula R¹XR² is directly and unambiguously derived, is evident, and iscontemplated, as if expressly listed herein and is from which the groupof positions for R¹XR² are readily envisioned as: D41, E63, P74, N91,S102, G106, A110, L111, V112, K118, I119, H120, G124, V125, D126, V127,S128, K129, E130, P131, M132, V134, S146, T149, T152, I153, K160, V161,V166, D167, Q270, G273, K348, N358, A363, C364. This is readily derivedas evident in the following that depicts the amino acid position foreach amino acid for substitution (corresponding to those of SEQ ID NO: 1and accepting the corresponding substitution). The above approachapplies to obtain a resulting R¹XR² formula for each templatepolypeptide herein, as well as for polypeptides sharing identity theretoas described herein.

GI: 387590701 MTNTQSAFFMPSVNLFGAGSVNEVGTRLADLGVKKALLVT D41AGLHGLGLSEKISSIIRAAGV E63 VSIFPKAEPN P74 T DKNVAEGLEAYNAE N91CDSIVTLGGGS S102 HDA G106 K AI A110L111V112 AANGG K118I119H120 DYEG124V12 5D126V127S128K129E130P131M132V134 PLIAINTT AGTG S146 EL T149 KFT152I153 ITDTER K160V161 KMA I V166D167 KHVTPTLSINDPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIKIISKYLPRAVANGKDI AREQMAFAQSLAGMAFNNAGLGYVHAIAHQ270 LG G273 FYNFPHGVCNAVLLPYVCRFNLISKVERYAEIAAFLGENVDGLSTYDAAEKAIKAIERMAKDLNIPKGFKELGA K348 E EDIETLAK N358 AMKD A363C364ALTNPRKPKLEEVIQII KNAM

In another example, the amino acids and positions for substitution R¹XR²in 387 amino acid template polypeptide YP_002138168.1 GI:197117741 (SEQID NO:17) from Geobacter bemidjiensis Bem with 52% global identity and71% similarity to SEQ ID NO:1 are: D43, S65, P76, G92, S104, A108, G112,M113, V114, H120, I121, R122, G126, V127, N128, K129, T130, T131, K132,P133, M134, P135, S148, T151, C154, I155, H162, V163, V168, D169, Q272,G275, K350, N360, A365, C366. The following, readily derivable from thetables herein, indicates these positions.

GI: 197117741 MALGEQTYGFYIPTVSLMGIGSAKETGGQIKALGASKALI VT D43KGLSAMGVADKIKSQVEEAGV S65 AVIFDGAEPN P76 TDINVHDGVKVYQDN G92 CDAIISLGGGSS104 HDC A 108 KGI G112M113V114 IGNGG H120I121R122 DLE G126V127N128K129T130T131K132P133M134 P 135 AFVA INTTAGTA S148 EM T151 RFC154I155 ITNTDT H162V16 3 KMAI V168D169 WRCTPNVAINDPLLMVGKPAALTAATGMDALTHAVEAYVSTIATPITDACAIKAIELIAEFLSKAVANGEDLEARDKMAYAEYLAGMAFNNASLGYVHSMAH Q272 LG G275FYNLPHGVCNAILLPAVSQYNLIACPKRF ADIAKALGENIDGLSVTEAGQKAIDRIRTLSASIGIPTGLKALNV K350 EADLTIMAE N360 AKKD A365C366 QF TNPRKATLEQVVQIFKDAM

Table 11 provides amino acid sequences of target polypeptides, havingunderlined target amino acids for substitution with the variant aminoacids generated in the Bacillus methanolicus MGA3 (2315A) variants. Itis understood that upon replacement of amino acid in the target sequence(with a variant amino acid from the corresponding location in the 2315Avariant), the substituted target sequence can be considered a “templatesequence,” useful in some embodiments for the further screening ofpolypeptides sequences for substitution.

Table 11 also illustrates a consensus of the templates of 60% or betteridentity to SEQ ID NO: 1 with positions for substitution indicated byunderlining. Non-underlined positions are not required for substitutionand, in embodiments, remain constant (identical across all templates).These positions can be tolerant to change by selection from at leastamongst the wild-type alternatives indicated at a specific position, andtolerant sites for substitution with the substitutions at the variantamino acid positions.

Site-directed mutagenesis or sequence alteration (e.g., site-specificmutagenesis or oligonucleotide-directed) can be used to make specificchanges to a target alcohol dehydrogenase DNA sequence to provide avariant DNA sequence encoding alcohol dehydrogenase with the desiredamino acid substitution. As a general matter, an oligonucleotide havinga sequence that provides a codon encoding the variant amino acid isused. Alternatively, artificial gene sequence of the entire codingregion of the variant alcohol dehydrogenase DNA sequence can beperformed as preferred alcohol dehydrogenases targeted for substitutionare generally less than 400 amino acids long.

Exemplary techniques using mutagenic oligonucleotides for generation ofa variant ADH sequence include the Kunkel method which may utilize anADH gene sequence placed into a phagemid. The phagemid in E. coliproduces ADH ssDNA which is template for mutagenesis using anoligonucleotide which is primer extended on the template.

Depending on the restriction enzyme sites flanking a location ofinterest in the ADH DNA, cassette mutagenesis may be used to create avariant sequence of interest. For cassette mutagenesis, a DNA fragmentis synthesized inserted into a plasmid, cleaved with a restrictionenzyme, and and then subsequently ligated to a pair of complementaryoligonucleotides containing the ADH variant mutation The restrictionfragments of the plasmid and oligonucleotide can be ligated to oneanother.

Another technique that can be used to generate the variant ADH sequenceis PCR site directed mutagensis. Mutageneic oligonucleotide primers areused to introduce the desired mutation and to provide a PCR fragmentcarrying the mutated sequence. Additional oligonucleotides may be usedto extend the ends of the mutated fragment to provide restriction sitessuitable for restriction enzyme digestion and insertion into the gene.

Commercial kits for site-directed mutagenesis techniques are alsoavailable. For example, the Quikchange™ kit uses complementary mutagenicprimers to PCR amplify a gene region using a high-fidelitynon-strand-displacing DNA polymerase such as pfu polymerase. Thereaction generates a nicked, circular DNA which is relaxed. The templateDNA is eliminated by enzymatic digestion with a restriction enzyme suchas DpnI which is specific for methylated DNA.

An expression vector or vectors can be constructed to include one ormore variant ADH encoding nucleic acids as exemplified herein operablylinked to expression control sequences functional in the host organism.Expression vectors applicable for use in the microbial host organismsprovided include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

The term “exogenous” is intended to mean that the referenced molecule orthe referenced activity is introduced into the host microbial organism.The molecule can be introduced, for example, by introduction of anencoding nucleic acid into the host genetic material such as byintegration into a host chromosome or as non-chromosomal geneticmaterial such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid canutilize either or both a heterologous or homologous encoding nucleicacid.

It is understood that when more than one exogenous nucleic acid isincluded in a microbial organism, the more than one exogenous nucleicacids refers to the referenced encoding nucleic acid or biosyntheticactivity, as discussed above. It is further understood, as disclosedherein, that more than one exogenous nucleic acids can be introducedinto the host microbial organism on separate nucleic acid molecules, onpolycistronic nucleic acid molecules, or a combination thereof, andstill be considered as more than one exogenous nucleic acid. Forexample, as disclosed herein a microbial organism can be engineered toexpress two or more exogenous nucleic acids encoding a desired pathwayenzyme or protein. In the case where two exogenous nucleic acidsencoding a desired activity are introduced into a host microbialorganism, it is understood that the two exogenous nucleic acids can beintroduced as a single nucleic acid, for example, on a single plasmid,on separate plasmids, can be integrated into the host chromosome at asingle site or multiple sites, and still be considered as two exogenousnucleic acids. Similarly, it is understood that more than two exogenousnucleic acids can be introduced into a host organism in any desiredcombination, for example, on a single plasmid, on separate plasmids, canbe integrated into the host chromosome at a single site or multiplesites, and still be considered as two or more exogenous nucleic acids,for example three exogenous nucleic acids. Thus, the number ofreferenced exogenous nucleic acids or biosynthetic activities refers tothe number of encoding nucleic acids or the number of biosyntheticactivities, not the number of separate nucleic acids introduced into thehost organism.

Exogenous variant ADH-encoding nucleic acid sequences can be introducedstably or transiently into a host cell using techniques well known inthe art including, but not limited to, conjugation, electroporation,chemical transformation, transduction, transfection, and ultrasoundtransformation. Optionally, for exogenous expression in E. coli or otherprokaryotic cells, some nucleic acid sequences in the genes or cDNAs ofeukaryotic nucleic acids can encode targeting signals such as anN-terminal mitochondrial or other targeting signal, which can be removedbefore transformation into prokaryotic host cells, if desired. Forexample, removal of a mitochondrial leader sequence led to increasedexpression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338(2005)). For exogenous expression in yeast or other eukaryotic cells,genes can be expressed in the cytosol without the addition of leadersequence, or can be targeted to mitochondrion or other organelles, ortargeted for secretion, by the addition of a suitable targeting sequencesuch as a mitochondrial targeting or secretion signal suitable for thehost cells. Thus, it is understood that appropriate modifications to anucleic acid sequence to remove or include a targeting sequence can beincorporated into an exogenous nucleic acid sequence to impart desirableproperties. Furthermore, genes can be subjected to codon optimizationwith techniques well known in the art to achieve optimized expression ofthe proteins.

The terms “microbial,” “microbial organism” or “microorganism” areintended to mean any organism that exists as a microscopic cell that isincluded within the domains of archaea, bacteria or eukarya. Therefore,the term is intended to encompass prokaryotic or eukaryotic cells ororganisms having a microscopic size and includes bacteria, archaea andeubacteria of all species as well as eukaryotic microorganisms such asyeast and fungi. The term also includes cell cultures of any speciesthat can be cultured for the production of a biochemical.

The term “isolated” when used in reference to a microbial organism isintended to mean an organism that is substantially free of at least onecomponent as the referenced microbial organism is found in nature. Theterm includes a microbial organism that is removed from some or allcomponents as it is found in its natural environment. The term alsoincludes a microbial organism that is removed from some or allcomponents as the microbial organism is found in non-naturally occurringenvironments.

In some aspects the ADH variant gene is introduced into a cell with agene disruption. The term “gene disruption,” or grammatical equivalentsthereof, is intended to mean a genetic alteration that renders theencoded gene product inactive or attenuated. The genetic alteration canbe, for example, deletion of the entire gene, deletion of a regulatorysequence required for transcription or translation, deletion of aportion of the gene which results in a truncated gene product, or by anyof various mutation strategies that inactivate or attenuate the encodedgene product. One particularly useful method of gene disruption iscomplete gene deletion because it reduces or eliminates the occurrenceof genetic reversions. The phenotypic effect of a gene disruption can bea null mutation, which can arise from many types of mutations includinginactivating point mutations, entire gene deletions, and deletions ofchromosomal segments or entire chromosomes. Specific antisense nucleicacid compounds and enzyme inhibitors, such as antibiotics, can alsoproduce null mutant phenotype, therefore being equivalent to genedisruption.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, microorganismsmay have genetic modifications to nucleic acids encoding metabolicpolypeptides, or functional fragments thereof. Exemplary metabolicmodifications are disclosed herein.

The microorganisms provided herein can contain stable geneticalterations, which refers to microorganisms that can be cultured forgreater than five generations without loss of the alteration. Generally,stable genetic alterations include modifications that persist greaterthan 10 generations, particularly stable modifications will persist morethan about 25 generations, and more particularly, stable geneticmodifications will be greater than 50 generations, includingindefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

A variety of microorganism may be suitable for the incorporating thevariant ADH, optionally with one or more other transgenes Such organismsinclude both prokaryotic and eukaryotic organisms including, but notlimited to, bacteria, including archaea and eubacteria, and eukaryotes,including yeast, plant, insect, animal, and mammal, including human.Exemplary species are reported in U.S. application Ser. No. 13/975,678(filed Aug. 26, 2013), which is incorporated herein by reference, andinclude, for example, Escherichia coli, Saccharomyces cerevisiae,Saccharomyces kluyveri, Candida boidinii, Clostridium kluyveri,Clostridium acetobutylicum, Clostridium beijerinckii, Clostridiumsaccharoperbutylacetonicum, Clostridium perfringens, Clostridiumdifficile, Clostridium botulinum, Clostridium tyrobutyricum, Clostridiumtetanomorphum, Clostridium tetani, Clostridium propionicum, Clostridiumaminobutyricum, Clostridium subterminale, Clostridium sticklandii,Ralstonia eutropha, Mycobacterium bovis, Mycobacterium tuberculosis,Porphyromonas gingivalis, Arabidopsis thaliana, Thermus thermophilus,Pseudomonas species, including Pseudomonas aeruginosa, Pseudomonasputida, Pseudomonas stutzeri, Pseudomonas fluorescens, Homo sapiens,Oryctolagus cuniculus, Rhodobacter spaeroides, Thermoanaerobacterbrockii, Metallosphaera sedula, Leuconostoc mesenteroides, Chloroflexusaurantiacus, Roseiflexus castenholzii, Erythrobacter, Simmondsiachinensis, Acinetobacter species, including Acinetobacter calcoaceticusand Acinetobacter baylyi, Porphyromonas gingivalis, Sulfolobus tokodaii,Sulfolobus solfataricus, Sulfolobus acidocaldarius, Bacillus subtilis,Bacillus cereus, Bacillus megaterium, Bacillus brevis, Bacillus pumilus,Rattus norvegicus, Klebsiella pneumonia, Klebsiella oxytoca, Euglenagracilis, Treponema denticola, Moorella thermoacetica, Thermotogamaritima, Halobacterium salinarum, Geobacillus stearothermophilus,Aeropyrum pernix, Sus scrofa, Caenorhabditis elegans, Corynebacteriumglutamicum, Acidaminococcus fermentans, Lactococcus lactis,Lactobacillus plantarum, Streptococcus thermophilus, Enterobacteraerogenes, Candida, Aspergillus terreus, Pedicoccus pentosaceus,Zymomonas mobilus, Acetobacter pasteurians, Kluyveromyces lactis,Eubacterium barkeri, Bacteroides capillosus, Anaerotruncus colihominis,Natranaerobius thermophilusm, Campylobacter jejuni, Haemophilusinfluenzae, Serratia marcescens, Citrobacter amalonaticus, Myxococcusxanthus, Fusobacterium nuleatum, Penicillium chrysogenum, marine gammaproteobacterium, butyrate producing bacterium, Nocardia iowensis,Nocardia farcinica, Streptomyces griseus, Schizosaccharomyces pombe,Geobacillus thermoglucosidasius, Salmonella typhimurium, Vibrio cholera,Heliobacter pylori, Nicotiana tabacum, Oryza sativa, Haloferaxmediterranei, Agrobacterium tumefaciens, Achromobacter denitrificans,Fusobacterium nucleatum, Streptomyces clavuligenus, Acinetobacterbaumanii, Mus musculus, Lachancea kluyveri, Trichomonas vaginalis,Trypanosoma brucei, Pseudomonas stutzeri, Bradyrhizobium japonicum,Mesorhizobium loti, Bos taurus, Nicotiana glutinosa, Vibrio vulnificus,Selenomonas ruminantium, Vibrio parahaemolyticus, Archaeoglobusfulgidus, Haloarcula marismortui, Pyrobaculum aerophilum, Mycobacteriumsmegmatis MC2 155, Mycobacterium avium subsp. paratuberculosis K-10,Mycobacterium marinum M, Tsukamurella paurometabola DSM 20162, CyanobiumPCC7001, Dictyostelium discoideum AX4, as well as other exemplaryspecies disclosed herein or available as source organisms forcorresponding genes.

In certain embodiments, suitable organisms include Acinetobacterbaumannii Naval-82, Acinetobacter sp. ADP1, Acinetobacter sp. strainM-1, Actinobacillus succinogenes 130Z, Allochromatium vinosum DSM 180,Amycolatopsis methanolica, Arabidopsis thaliana, Atopobium parvulum DSM20469, Azotobacter vinelandii DJ, Bacillus alcalophilus ATCC 27647,Bacillus azotoformans LMG 9581, Bacillus coagulans 36D1, Bacillusmegaterium, Bacillus methanolicus MGA3, Bacillus methanolicus PB1,Bacillus methanolicus PB-1, Bacillus selenitireducens MLS10, Bacillussmithii, Bacillus subtilis, Burkholderia cenocepacia, Burkholderiacepacia, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderiastabilis, Burkholderia thailandensis E264, Burkholderiales bacteriumJoshi_001, Butyrate-producing bacterium L2-50, Campylobacter jejuni,Candida albicans, Candida boidinii, Candida methylica, Carboxydothermushydrogenoformans, Carboxydothermus hydrogenoformans Z-2901, Caulobactersp. AP07, Chloroflexus aggregans DSM 9485, Chloroflexus aurantiacusJ-10-fl, Citrobacter freundii, Citrobacter koseri ATCC BAA-895,Citrobacter youngae, Clostridium, Clostridium acetobutylicum,Clostridium acetobutylicum ATCC 824, Clostridium acidurici, Clostridiumaminobutyricum, Clostridium asparagiforme DSM 15981, Clostridiumbeijerinckii, Clostridium beijerinckii NCIMB 8052, Clostridium bolteaeATCC BAA-613, Clostridium carboxidivorans P7, Clostridium cellulovorans743B, Clostridium difficile, Clostridium hiranonis DSM 13275,Clostridium hylemonae DSM 15053, Clostridium kluyveri, Clostridiumkluyveri DSM 555, Clostridium ljungdahli, Clostridium ljungdahlii DSM13528, Clostridium methylpentosum DSM 5476, Clostridium pasteurianum,Clostridium pasteurianum DSM 525, Clostridium perfringens, Clostridiumperfringens ATCC 13124, Clostridium perfringens str. 13, Clostridiumphytofermentans ISDg, Clostridium saccharobutylicum, Clostridiumsaccharoperbutylacetonicum, Clostridium saccharoperbutylacetonicum N1-4,Clostridium tetani, Corynebacterium glutamicum ATCC 14067,Corynebacterium glutamicum R, Corynebacterium sp. U-96, Corynebacteriumvariabile, Cupriavidus necator N-1, Cyanobium PCC7001, Desulfatibacillumalkenivorans AK-01, Desulfitobacterium hafniense, Desulfitobacteriummetallireducens DSM 15288, Desulfotomaculum reducens Desulfovibrioafricanus str. Walvis Bay, Desulfovibrio fructosovorans JJ,Desulfovibrio vulgaris str. Hildenborough, Desulfovibrio vulgaris str.‘Miyazaki F’, Dictyostelium discoideum AX4, Escherichia coli,Escherichia coli K-12, Escherichia coli K-12 MG1655, Eubacterium halliiDSM 3353, Flavobacterium frigoris, Fusobacterium nucleatum subsp.polymorphum ATCC 10953, Geobacillus sp. Y4.1MC1, Geobacillusthemodenitrificans NG80-2, Geobacter bemidjiensis Bem, Geobactersulfurreducens, Geobacter sulfurreducens PCA, Geobacillusstearothermophilus DSM 2334, Haemophilus influenzae, Helicobacterpylori, Homo sapiens, Hydrogenobacter thermophilus, Hydrogenobacterthermophilus TK-6, Hyphomicrobium denitrificans ATCC 51888,Hyphomicrobium zavarzinii, Klebsiella pneumoniae, Klebsiella pneumoniaesubsp. pneumoniae MGH 78578, Lactobacillus brevis ATCC 367, Leuconostocmesenteroides, Lysinibacillus fusiformis, Lysinibacillus sphaericus,Mesorhizobium loti MAFF303099, Metallosphaera sedula, Methanosarcinaacetivorans, Methanosarcina acetivorans C2A, Methanosarcina barkeri,Methanosarcina mazei Tuc01, Methylobacter marinus, Methylobacteriumextorquens, Methylobacterium extorquens AM1, Methylococcus capsulatas,Methylomonas aminofaciens, Moorella thermoacetica, Mycobacter sp. strainJC1 DSM 3803, Mycobacterium avium subsp. paratuberculosis K-10,Mycobacterium bovis BCG, Mycobacterium gastri, Mycobacterium marinum M,Mycobacterium smegmatis, Mycobacterium smegmatis MC2 155, Mycobacteriumtuberculosis, Nitrosopumilus salaria BD31, Nitrososphaera gargensisGa9.2, Nocardia farcinica IFM 10152, Nocardia iowensis (sp. NRRL 5646),Nostoc sp. PCC 7120, Ogataea angusta, Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1), Paenibacillus peoriae KCTC 3763, Paracoccusdenitrificans, Penicillium chrysogenum, Photobacterium profundum 3TCK,Phytofermentans ISDg, Pichia pastoris, Picrophilus torridus DSM9790,Porphyromonas gingivalis, Porphyromonas gingivalis W83, Pseudomonasaeruginosa PA01, Pseudomonas denitrificans, Pseudomonas knackmussii,Pseudomonas putida, Pseudomonas sp, Pseudomonas syringae pv. syringaeB728a, Pyrobaculum islandicum DSM 4184, Pyrococcus abyssi, Pyrococcusfuriosus, Pyrococcus horikoshii OT3, Ralstonia eutropha, Ralstoniaeutropha H16, Rhodobacter capsulatus, Rhodobacter sphaeroides,Rhodobacter sphaeroides ATCC 17025, Rhodopseudomonas palustris,Rhodopseudomonas palustris CGA009, Rhodopseudomonas palustris DX-1,Rhodospirillum rubrum, Rhodospirillum rubrum ATCC 11170, Ruminococcusobeum ATCC 29174, Saccharomyces cerevisiae, Saccharomyces cerevisiaeS288c, Salmonella enterica, Salmonella enterica subsp. enterica serovarTyphimurium str. LT2, Salmonella enterica typhimurium, Salmonellatyphimurium, Schizosaccharomyces pombe, Sebaldella termitidis ATCC33386, Shewanella oneidensis MR-1, Sinorhizobium meliloti 1021,Streptomyces coelicolor, Streptomyces griseus subsp. griseus NBRC 13350,Sulfolobus acidocalarius, Sulfolobus solfataricus P-2, Synechocystisstr. PCC 6803, Syntrophobacter fumaroxidans, Thauera aromatica,Thermoanaerobacter sp. X514, Thermococcus kodakaraensis, Thermococcuslitoralis, Thermoplasma acidophilum, Thermoproteus neutrophilus,Thermotoga maritima, Thiocapsa roseopersicina, Tolumonas auensis DSM9187, Trichomonas vaginalis G3, Trypanosoma brucei, Tsukamurellapaurometabola DSM 20162, Vibrio cholera, Vibrio harveyi ATCC BAA-1116,Xanthobacter autotrophicus Py2, Yersinia intermedia, or Zea mays.

In some aspects the variant ADH gene is introduced into a cellengineered with increased of levels of 1,4-butanediol (BDO) orhydroxylbutyrate (4-HB) biosynthetic capability, those skilled in theart will understand with applying the teaching and guidance providedherein to a particular species that the identification of metabolicmodifications can include identification and inclusion or inactivationof orthologs. To the extent that paralogs and/or nonorthologous genedisplacements are present in the referenced microorganism that encode anenzyme catalyzing a similar or substantially similar metabolic reaction,those skilled in the art also can utilize these evolutionally relatedgenes.

With the complete genome sequence available for now more than 550species (with more than half of these available on public databases suchas the NCBI), including 395 microorganism genomes and a variety ofyeast, fungi, plant, and mammalian genomes, the identification of genesencoding the requisite BDO or 4-HB biosynthetic pathway as well as otherknown biosynthetic pathways for 1,3-butanediol (13BDO), butadiene,6-amino caproic acid (6ACA), hexamethyldiamine (HMDA), adipic acid orderivatives thereof, croytl alcohol, methyl vinyl carbinol,3-buten-1-ol, succinic acid or derivatives thereof, n-propanol,isopropanol, propylene, methacrylic acid or derivatives thereof,methanol metabolic and/or formaldehyde assimilation activity for one ormore genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations allowing biosynthesis of various target productsincluding 1,3-butanediol (13BDO), 1, 4-butanediol (BDO), 4-HB,butadiene, 6-amino caproic acid (6ACA), hexamethyldiamine (HMDA), adipicacid or derivatives thereof, croytl alcohol, methyl vinyl carbinol,3-buten-1-ol, succinic acid or derivatives thereof, n-propanol,isopropanol, propylene, methacrylic acid or derivatives thereof,metabolism of methanol and/or assimilation of formaldehyde describedherein with reference to a particular organism such as E. coli can bereadily applied to other microorganisms, including prokaryotic andeukaryotic organisms alike. Given the teachings and guidance providedherein, those skilled in the art will know that a metabolic alterationexemplified in one organism can be applied equally to other organisms.

Therefore, the engineered cell including the non-natural NAD⁺-dependentalcohol dehydrogenase, can include one or more genetic alterations, suchas inserted transgenes, deletions, attenuation, mutations, etc., desiredto increase levels of one or more intermediates or a product thereof,and include those genetic modifications as described in U.S. applicationSer. No. 13/975,678 (filed Aug. 26, 2013), which is incorporated hereinby reference.

Exemplary alcohol metabolic pathway gene(s), such as described in U.S.application Ser. No. 13/975,678, encode a protein selected from thegroup consisting of a), a formate dehydrogenase (EM8), a formaldehydeactivating enzyme (EM10), a formaldehyde dehydrogenase (EM11), aS-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependentformaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase(EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16), anyor more can be coexpressed with the non-natural NAD⁺-dependent alcoholdehydrogenase in the engineered cell.

Other exemplary alcohol metabolic pathway gene(s), such as described inU.S. application Ser. No. 13/975,678, encode an alcohol metabolicpathway gene(s) encoding a protein selected from the group consisting ofa succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate(4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), aphosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase(aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); asuccinate reductase (EB9), a succinyl-CoA reductase (alcohol forming)(EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoAsynthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoAreductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1),and a succinyl-CoA synthetase (EB2A), any or more can be coexpressedwith the non-natural NAD⁺-dependent alcohol dehydrogenase in theengineered cell.

Target products obtained from, and product pathways suitable forproducing in, host cells expressing the engineered NAD+-dependentmethanol or ethanol dehydrogenases described herein include thefollowing. Of particular interest are a target product obtained usingpyruvate and acetyl-CoA as entry point or precursor to its productpathway(s), in part because the methanol metabolic pathway using thenovel enzymes enables fixing the carbon of methanol into pathways topyruvate and acetyl-CoA. Target products include (a) 1,4-butanediol andintermediates thereto, such as 4-hydroxybutanoic acid(4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), (b) butadiene andintermediates thereto, such as 1,4-butanediol, 1,3-butanediol, crotylalcohol, 3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol, (c)1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate,crotyl alcohol or 3-buten-1-ol, (d) adipate, 6-aminocaproic acid,caprolactam, hexamethylenediamine and levulinic acid and theirintermediates, e.g. 4-aminobutyryl-CoA, (e) methacrylic acid(2-methyl-2-propenoic acid) and its esters known collectively asmethacrylates, such as methyl methacrylate, methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and theirintermediates, (f) 1,2-propanediol (propylene glycol), n-propanol,1,3-propanediol and glycerol, and their intermediates and (g) succinicacid and intermediates thereto.

1,4-butanediol and intermediates thereto, such as 4-hydroxybutanoic acid(4-hydroxybutanoate, 4-hydroxybutyrate, 4-HB), are target products thatcan be made by co-expressing the novel alcohol dehydrogenases describedherein with a product pathway described herein as well as in thefollowing documents. Suitable product pathways and enzymes, methods forscreening and methods for isolating are found herein as well as in thefollowing documents, incorporated herein by reference: WO2008115840A2published 25 Sep. 2008 entitled Compositions and Methods for theBiosynthesis of 1, 4-Butanediol and Its Precursors; WO2010141780A1published 9 Dec. 2010 entitled Process of Separating Components of AFermentation Broth; WO2010141920A2 published 9 Dec. 2010 entitledMicroorganisms for the Production of 1, 4-Butanediol and RelatedMethods; WO2010030711A2 published 18 Mar. 2010 entitled Microorganismsfor the Production of 1, 4-Butanediol; WO2010071697A1 published 24 Jun.2010 Microorganisms and Methods for Conversion of Syngas and OtherCarbon Sources to Useful Products; WO2009094485A1 published 30 Jul. 2009Methods and Organisms for Utilizing Synthesis Gas or Other GaseousCarbon Sources and Methanol; WO2009023493A1 published 19 Feb. 2009entitled Methods and Organisms for the Growth-Coupled Production of 1,4-Butanediol; WO2008115840A2 published 25 Sep. 2008 entitledCompositions and Methods for the Biosynthesis of 1,4-Butanediol and ItsPrecursors; and International Application No. PCT/US13/56725 filed 27Aug. 2013 entitled Microorganisms an Methods for Enhancing theAvailability of Reducing Equivalents in the Presence of Methanol, andfor Producing 1,4-Butanediol Related Thereto.

Butadiene and intermediates thereto, such as 1,4-butanediol,1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and3-buten-1-ol, are target products that can be made by co-expressing thenovel alcohol dehydrogenases described herein with a product pathwaydescribed in the following documents. In addition to direct fermentationto produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol,3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol can be separated,purified (for any use), and then dehydrated to butadiene in a secondstep involving metal-based catalysis. Suitable product pathways andenzymes, methods for screening and methods for isolating are found inthe following documents, incorporated herein by reference:WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms andMethods for the Biosynthesis of Butadiene; WO2012018624A2 published 9Feb. 2012 entitled Microorganisms and Methods for the Biosynthesis ofAromatics, 2,4-Pentadienoate and 1,3-Butadiene; WO2011140171A2 published10 Nov. 2011 entitled Microorganisms and Methods for the Biosynthesis ofButadiene; WO2013040383A1 published 21 Mar. 2013 entitled Microorganismsand Methods for Producing Alkenes; WO2012177710A1 published 27 Dec. 2012entitled Microorganisms for Producing Butadiene and Methods Relatedthereto; WO2012106516A1 published 9 Aug. 2012 entitled Microorganismsand Methods for the Biosynthesis of Butadiene; WO2013028519A1 published28 Feb. 2013 entitled Microorganisms and Methods for Producing2,4-Pentadienoate, Butadiene, Propylene, 1,3-Butanediol and RelatedAlcohols; and U.S. Ser. No. 61/799,255 filed 15 Mar. 2013.

1,3-butanediol and intermediates thereto, such as 2,4-pentadienoate,crotyl alcohol or 3-buten-1-ol, are target products that can be made byco-expressing the novel alcohol dehydrogenases described herein with aproduct pathway described herein as well as in the following documents.Suitable product pathways and enzymes, methods for screening and methodsfor isolating are found herein as well as in the following documents,incorporated herein by reference: WO2011071682A1 published 16 Jun. 2011entitled Methods and Organisms for Converting Synthesis Gas or OtherGaseous Carbon Sources and Methanol to 1, 3-Butanediol; WO2011031897Apublished 17 Mar. 2011 entitled Microorganisms and Methods for theCo-Production of Isopropanol with Primary Alcohols, Diols and Acids;WO2010127319A2 published 4 Nov. 2010 entitled Organisms for theProduction of 1,3-Butanediol; WO2013071226A1 published 16 May 2013entitled Eukaryotic Organisms and Methods for Increasing theAvailability of Cytosolic Acetyl-CoA, and for Producing 1,3-Butanediol;WO2013028519A1 published 28 Feb. 2013 entitled Microorganisms andMethods for Producing 2,4-Pentadienoate, Butadiene, Propylene,1,3-Butanediol and Related Alcohols; WO2013036764A1 published 14 Mar.2013 entitled Eukaryotic Organisms and Methods for Producing1,3-Butanediol; WO2013012975A1 published 24 Jan. 2013 entitled Methodsfor Increasing Product Yields; WO2012177619A2 published 27 Dec. 2012entitled Microorganisms for Producing 1, 3-Butanediol and MethodsRelated Thereto; and U.S. Ser. No. 61/799,255 filed 15 Mar. 2013.

Adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine andlevulinic acid, and their intermediates, e.g. 4-aminobutyryl-CoA, aretarget products, useful for example for making nylon polymers, that canbe made by co-expressing the novel alcohol dehydrogenases describedherein with a product pathway described herein as well as in thefollowing documents. Suitable product pathways and enzymes, methods forscreening and methods for isolating are found herein as well as in thefollowing documents, incorporated herein by reference: WO2010129936A1published 11 Nov. 2010 entitled Microorganisms and Methods for theBiosynthesis of Adipate, Hexamethylenediamine and 6-Aminocaproic Acid;WO2013012975A1 published 24 Jan. 2013 entitled Methods for IncreasingProduct Yields; WO2012177721A1 published 27 Dec. 2012 entitledMicroorganisms for Producing 6-Aminocaproic Acid; WO2012099621A1published 26 Jul. 2012 entitled Methods for Increasing Product Yields;and application U.S. Ser. No. 61/766,620 filed 19 Feb. 2013 entitledMicroorganisms an Methods for Enhancing the Availability of ReducingEquivalents in the Presence of Methanol, and for Producing Adipate,6-Aminocaproate, Hexamethylenediamine or Caprolactam Related Thereto.

Methacrylic acid (2-methyl-2-propenoic acid; used in the preparation ofits esters known collectively as methacrylates, such as methylmethacrylate, which is used most notably in the manufacture ofpolymers), methacrylate ester such as methyl methacrylate,3-hydroxyisobutyrate and/or 2-hydroxyisobutyrate and their intermediatesare target products, useful for example for making polymers, that can bemade by co-expressing the novel alcohol dehydrogenases described hereinwith a product pathway described herein as well as in the followingdocuments. Suitable product pathways and enzymes, methods for screeningand methods for isolating are found herein as well as in the followingdocuments, incorporated herein by reference: WO2012135789A2 published 4Oct. 2012 entitled Microorganisms for Producing Methacrylic Acid andMethacrylate Esters and Methods Related Thereto; WO2009135074A2published 5 Nov. 2009 entitled Microorganisms for the Production ofMethacrylic Acid; and application U.S. Ser. No. 61/766,660 filed 19 Feb.2013 entitled Microorganisms an Methods for Enhancing the Availabilityof Reducing Equivalents in the Presence of Methanol, and for Producing3-Hydroxyisobutyate or Methacrylic Acid Related Thereto.

1,2-propanediol (propylene glycol), n-propanol, 1,3-propanediol andglycerol, and their intermediates are target products, useful forexample for making polymers, that can be made by co-expressing the novelalcohol dehydrogenases described herein with a product pathway describedherein as well as in the following documents. Suitable product pathwaysand enzymes, methods for screening and methods for isolating are foundherein as well as in the following documents, incorporated herein byreference: WO2009111672A1 published 9 Nov. 2009 entitled Primary AlcoholProducing Organisms; WO2011031897A1 17 Mar. 2011 entitled Microorganismsand Methods for the Co-Production of Isopropanol with Primary Alcohols,Diols and Acids; WO2012177599A2 published 27 Dec. 2012 entitledMicroorganisms for Producing N-Propanol 1, 3-Propanediol, 1,2-Propanediol or Glycerol and Methods Related Thereto; and applicationU.S. Ser. No. 61/766,635 filed 19 Feb. 2013 entitled Microorganisms anMethods for Enhancing the Availability of Reducing Equivalents in thePresence of Methanol, and for Producing 1,2-Propanediol, n-Propanol,1,3-Propanediol, or Glycerol Related Thereto.

Succinic acid and intermediates thereto (useful to produce productsincluding polymers, e.g. PBS, 1,4-butanediol, tetrahydrofuran,pyrrolidone, solvents, paints, deicers, plastics, fuel additives,fabrics, carpets, pigments, and detergents) are target products that canbe made by co-expressing the novel alcohol dehydrogenases describedherein with a product pathway described herein as well as in thefollowing documents. Suitable product pathways and enzymes, methods forscreening and methods for isolating are found herein as well as in thefollowing documents, incorporated herein by reference: EP1937821A2published 2 Jul. 2008 entitled Methods and Organisms for theGrowth-Coupled Production of Succinate; and application U.S. Ser. No.61/766,635 filed 19 Feb. 2013 entitled Microorganisms and Methods forEnhancing the Availability of Reducing Equivalents in the Presence ofMethanol, and for Producing Succinate Related Thereto.

Target products obtained from, and product pathways suitable forproducing in, host cells co-expressing the engineered NAD+-dependentmethanol or ethanol dehydrogenases described herein include thefollowing.

Butadiene and intermediates thereto, such as 1,4-butanediol,1,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) and3-buten-1-ol, are target products that can be made by co-expressing thenovel alcohol dehydrogenases described herein with a product pathwaydescribed in the following documents. In addition to direct fermentationto produce butadiene, 1,3-butanediol, 1,4-butanediol, crotyl alcohol,3-buten-2-ol (methyl vinyl carbinol) and 3-buten-1-ol can be separated,purified (for any use), and then dehydrated to butadiene in a secondstep involving metal-based catalysis. Suitable product pathways andenzymes, methods for screening and methods for isolating are found in:WO2011140171A2 published 10 Nov. 2011 entitled Microorganisms andMethods for the Biosynthesis of Butadiene; WO2012018624A2 published 9Feb. 2012 entitled Microorganisms and Methods for the Biosynthesis ofAromatics, 2, 4-Pentadienoate and 1, 3-Butadiene; O2011140171A2published 10 Nov. 2011 entitled Microorganisms and Methods for theBiosynthesis of Butadiene; WO2013040383A1 published 21 Mar. 2013entitled Microorganisms and Methods for Producing Alkenes;WO2012177710A1 published 27 Dec. 2012 entitled Microorganisms forProducing Butadiene and Methods Related thereto; WO2012106516A1published 9 Aug. 2012 entitled Microorganisms and Methods for theBiosynthesis of Butadiene; WO2013028519A1 published 28 Feb. 2013entitled Microorganisms and Methods for Producing 2,4-Pentadienoate,Butadiene, Propylene, 1,3-Butanediol and Related Alcohols; and U.S. Ser.No. 61/799,255 filed 15 Mar. 2013.

In some embodiments, the disclosure provides organisms comprising a MDHvariant and that are engineered to improve the availability of reducingequivalents or utilizing formaldehyde resulting from methanol via aformaldehyde assimilation pathway (FAB), which can be used for theproduction of target product molecules. It will be recognized by oneskilled in the art that any product molecule that utilizes reducingequivalents in its production can exhibit enhanced production throughother biosynthetic pathways.

BDO is a valuable chemical for the production of high performancepolymers, solvents, and fine chemicals. It is the basis for producingother high value chemicals such as tetrahydrofuran (THF) andgamma-butyrolactone (GBL). The value chain is comprised of three mainsegments including: (1) polymers, (2) THF derivatives, and (3) GBLderivatives. In the case of polymers, BDO is a comonomer forpolybutylene terephthalate (PBT) production. PBT is a medium performanceengineering thermoplastic used in automotive, electrical, water systems,and small appliance applications. Conversion to THF, and subsequently topolytetramethylene ether glycol (PTMEG), provides an intermediate usedto manufacture spandex products such as LYCRA® fibers. PTMEG is alsocombined with BDO in the production of specialty polyester ethers(COPE). COPEs are high modulus elastomers with excellent mechanicalproperties and oil/environmental resistance, allowing them to operate athigh and low temperature extremes. PTMEG and BDO also make thermoplasticpolyurethanes processed on standard thermoplastic extrusion,calendaring, and molding equipment, and are characterized by theiroutstanding toughness and abrasion resistance. The GBL produced from BDOprovides the feedstock for making pyrrolidones, as well as serving theagrochemical market. The pyrrolidones are used as high performancesolvents for extraction processes of increasing use, including forexample, in the electronics industry and in pharmaceutical production.Accordingly, provided herein is bioderived BDO produced according to themethods described herein and biobased products comprising or obtainedusing the bioderived BDO.

In numerous engineered pathways, realization of maximum product yieldsbased on carbohydrate feedstock is hampered by insufficient reducingequivalents or by loss of reducing equivalents to byproducts. Methanolis a relatively inexpensive organic feedstock that can be used togenerate reducing equivalents by employing one or more methanolmetabolic enzymes as shown in FIG. 3a . The reducing equivalentsproduced by the metabolism of methanol can then be used to power theglucose to BDO production pathways, for example, as shown in FIG. 2.

IN FIG. 2, the organism comprises at least one exogenous nucleic acidencoding a BDOPE expressed in a sufficient amount to produce BDO. Incertain embodiments, the BDOPE is selected from the group consisting ofa succinyl-CoA transferase (EB1) or a succinyl-CoA synthetase (EB2A) (orsuccinyl-CoA ligase); a succinyl-CoA reductase (aldehyde forming) (EB3);a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4); a 4-HB kinase (EB5); aphosphotrans-4-hydroxybutyrylase (EB6); a 4-hydroxybutyryl-CoA reductase(aldehyde forming) (EB7); a 1,4-butanediol dehydrogenase (EB8); asuccinate reductase (EB9); a succinyl-CoA reductase (alcohol forming)(EB10); a 4-hydroxybutyryl-CoA transferase (EB11) or a4-hydroxybutyryl-CoA synthetase (EB12); a 4-HB reductase (EB13); a4-hydroxybutyryl-phosphate reductase (EB14); and a 4-hydroxybutyryl-CoAreductase (alcohol forming) (EB15).

Enzymes, genes and methods for engineering pathways from succinate andsuccinyl-CoA to various products, such as BDO, into a microorganism, arenow known in the art (see, e.g., U.S. Publ. No. 2011/0201089). A set ofBDOPEs represents a group of enzymes that can convert succinate to BDOas shown in FIG. 2. The additional reducing equivalents obtained fromthe MDH pathway, as disclosed herein, improve the yields of all theseproducts when utilizing carbohydrate-based feedstock. For example, BDOcan be produced from succinyl-CoA via previously disclosed pathways (seefor example, Burk et al., WO 2008/115840). Exemplary enzymes for theconversion succinyl-CoA to BDO include EB3 (FIG. 2, Step B), EB4 (FIG.2, Step C), EB5 (FIG. 2, Step D), EB6 (FIG. 2, Step E), EB7 (FIG. 2,Step F), EB8 (FIG. 2, Step G), EB10 (FIG. 1, Step I), EB11 (FIG. 2, StepJ), EB12 (FIG. 2, Step J), EB14 (FIG. 2, Step L), EB13 (FIG. 2, Step K),and EB15 (FIG. 2, Step M). EB9 (FIG. 2, Step H) can be additionallyuseful in converting succinate directly to the BDOP intermediate,succinate semialdehyde.

The maximum theoretical yield of BDO via the pathway shown in FIG. 2supplemented with the reactions of the oxidative TCA cycle (e.g.,citrate synthase, aconitase, isocitrate dehydrogenase,alpha-ketoglutarate dehydrogenase) is 1.09 mol/mol.1C₆H₁₂O₆→1.09C₄H₁₀O₂+1.64CO₂+0.55H₂O

When both feedstocks of sugar and methanol are available, the methanolcan be utilized to generate reducing equivalents by employing one ormore of the enzymes shown in FIG. 1. The reducing equivalents generatedfrom methanol can be utilized to power the glucose to BDO productionpathways, e.g., as shown in FIG. 2. Theoretically, all carbons inglucose will be conserved, thus resulting in a maximal theoretical yieldto produce BDO from glucose at 2 mol BDO per mol of glucose under eitheraerobic or anaerobic conditions as shown in FIG. 2:10CH₃OH+3C₆H₁₂O₆=6C₄H₁₀O₂+8H₂O+4CO₂

In a similar manner, the maximum theoretical yields of succinate and4-HB can reach 2 mol/mol glucose using the reactions shown in FIGS. 1and 2.C₆H₁₂O₆+0.667CH₃OH+1.333CO₂→2C₄H₆O₄+1.333H₂OC₆H₁₂O₆+2CH₃OH→2C₄H₈O₃+2H₂O

In other embodiments, the organism having a MDH protein, either alone orin combination with a BDOP, as provided herein, may further comprises aformaldehyde assimilation pathway (FAP) that utilizes formaldehyde,e.g., obtained from the oxidation of methanol, in the formation ofintermediates of certain central metabolic pathways that can be used,for example, in the formation of biomass. In certain embodiments, theorganism further comprises a FAP, wherein said organism comprises atleast one exogenous nucleic acid encoding a formaldehyde assimilationpathway enzyme (FAPE) expressed in a sufficient amount to produce anintermediate of glycolysis and/or a metabolic pathway that can be usedin the formation of biomass. In one embodiment, the FAPE is expressed ina sufficient amount to produce an intermediate of glycolysis. In anotherembodiment, the FAPE is expressed in a sufficient amount to produce anintermediate of a metabolic pathway that can be used in the formation ofbiomass. In some of the embodiments, the FAP comprises ahexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase(EF2), a dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4). Inone embodiment, the FAP comprises an EF1 and an EF2. In one embodiment,the intermediate is a H6P, a fructose-6-phosphate (F6P), or acombination thereof. In other embodiments, the FAP comprises an EF3 oran EF4. In one embodiment, the intermediate is a DHA, a DHA phosphate,or a combination thereof. In certain embodiments, the organism comprisestwo exogenous nucleic acids, each encoding a FAPE.

Also provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (e.g., as provided in FIG. 3A,step J) in the formation of intermediates of certain central metabolicpathways that can be used for the formation of biomass. One exemplaryFAP that can utilize formaldehyde produced from the oxidation ofmethanol (e.g., as provided in FIG. 3A) is shown in FIG. 3b , whichinvolves condensation of formaldehyde and D-ribulose-5-phosphate to formH6P by EF1 (FIG. 3b , step A). The enzyme can use Mg²⁺ or Mn²⁺ formaximal activity, although other metal ions are useful, and evennon-metal-ion-dependent mechanisms are contemplated. H6P is convertedinto F6P by EF2 (FIG. 3b , step B). Another exemplary pathway thatinvolves the detoxification and assimilation of formaldehyde producedfrom the oxidation of methanol (e.g., as provided in FIG. 3a ) is shownin FIG. 3c and proceeds through DHA. EF3 is a special transketolase thatfirst transfers a glycoaldehyde group from xylulose-5-phosphate toformaldehyde, resulting in the formation of DHA and G3P, which is anintermediate in glycolysis (FIG. 3c , step A). The DHA obtained from DHAsynthase is then further phosphorylated to form DHA phosphate by a DHAkinase (FIG. 3c , step B). DHAP can be assimilated into glycolysis andseveral other pathways. Rather than converting formaldehyde to formateand on to CO₂ off-gassed, the pathways provided in FIGS. 3b and 3c showthat carbon is assimilated, going into the final product.

Thus, in one embodiment, an organism having a MDH protein, either aloneor in combination with a BDOP, as provided herein, further comprises aFAP that utilizes formaldehyde, e.g., obtained from the oxidation ofmethanol, in the formation of intermediates of certain central metabolicpathways that can be used, for example, in the formation of biomass. Insome embodiments, the FAP comprises 3A or 3B, wherein 3A is an EF1 and3B is an EF2 In other embodiments, the FAP comprises 4A or 4B, wherein4A is an EF3 and 4B is an EF4. In certain embodiments, provided hereinis a organism having a MDH protein, wherein said organism comprises atleast one exogenous nucleic acid encoding an EM9 expressed in asufficient amount to enhance the availability of reducing equivalents inthe presence of methanol and/or expressed in a sufficient amount toconvert methanol to formaldehyde. In some embodiments, the organismcomprises at least one exogenous nucleic acid encoding an EM9 expressedin a sufficient amount to enhance the availability of reducingequivalents in the presence of methanol. In other embodiments, theorganism comprises at least one exogenous nucleic acid encoding an EM9expressed in a sufficient amount to convert methanol to formaldehyde. Insome embodiments, the microbial organism further comprises a FAP. Incertain embodiments, the organism further comprises at least oneexogenous nucleic acid encoding a FAPE expressed in a sufficient amountto produce an intermediate of glycolysis. In certain embodiments, theFAPE is selected from the group consisting of an EF1, an EF2, an EF3 andan EF4.

Exemplary enzymes suitable for the reactions described herein tometabolize methanol for either or both reducing equivalents or carboninclude the following, with respect to FIG. 3A, particularly as regardsto Steps J, L, I, G, H, M, N and O.

FIG. 3, Step G—Formate Hydrogen Lyase (EM15)

An EM15 enzyme can be employed to convert formate to carbon dioxide andhydrogen. An exemplary EM15 enzyme can be found in Escherichia coli. TheE. coli EM15 consists of hydrogenase 3 and formate dehydrogenase-H(Maeda et al., Appl Microbiol Biotechnol 77:879-890 (2007)). It isactivated by the gene product of fhlA. (Maeda et al., Appl MicrobiolBiotechnol 77:879-890 (2007)). The addition of the trace elements,selenium, nickel and molybdenum, to a fermentation broth has been shownto enhance EM15 activity (Soini et al., Microb. Cell Fact. 7:26 (2008)).Various hydrogenase 3, EM8 and transcriptional activator genes are shownbelow.

Protein GenBank ID GI number Organism hycA NP_417205 16130632Escherichia coli K-12 MG1655 hycB NP_417204 16130631 Escherichia coliK-12 MG1655 hycC NP_417203 16130630 Escherichia coli K-12 MG1655 hycDNP_417202 16130629 Escherichia coli K-12 MG1655 hycE NP_417201 16130628Escherichia coli K-12 MG1655 hycF NP_417200 16130627 Escherichia coliK-12 MG1655 hycG NP_417199 16130626 Escherichia coli K-12 MG1655 hycHNP_417198 16130625 Escherichia coli K-12 MG1655 hycI NP_417197 16130624Escherichia coli K-12 MG1655 fdhF NP_418503 16131905 Escherichia coliK-12 MG1655 fhlA NP_417211 16130638 Escherichia coli K-12 MG1655

An EM15 enzyme also exists in the hyperthermophilic archaeon,Thermococcus litoralis (Takacs et al., BMC. Microbiol 8:88 (2008)).

Protein GenBank ID GI number Organism mhyC ABW05543 157954626 mhyDABW05544 157954627 Thermococcus litoralis mhyE ABW05545 157954628Thermococcus litoralis myhF ABW05546 157954629 Thermococcus litoralismyhG ABW05547 157954630 Thermococcus litoralis myhH ABW05548 157954631Thermococcus litoralis fdhA AAB94932 2746736 Thermococcus litoralis fdhBAAB94931 157954625 Thermococcus litoralis

Additional EM15 systems have been found in Salmonella typhimurium,Klebsiella pneumoniae, Rhodospirillum rubrum, Methanobacteriumformicicum (Vardar-Schara et al., Microbial Biotechnology 1:107-125(2008)).

FIG. 3, Step H—Hydrogenase (EM16)

Hydrogenase enzymes can convert hydrogen gas to protons and transferelectrons to acceptors such as ferredoxins, NAD+, or NADP+. Ralstoniaeutropha H16 uses hydrogen as an energy source with oxygen as a terminalelectron acceptor. Its membrane-bound uptake [NiFe]-hydrogenase is an“O2-tolerant” EM16 (Cracknell, et al. Proc Nat Acad Sci, 106(49)20681-20686 (2009)) that is periplasmically-oriented and connected tothe respiratory chain via a b-type cytochrome (Schink and Schlegel,Biochim. Biophys. Acta, 567, 315-324 (1979); Bernhard et al., Eur. J.Biochem. 248, 179-186 (1997)). R. eutropha also contains an O₂-tolerantsoluble EM16 encoded by the Hox operon which is cytoplasmic and directlyreduces NAD+ at the expense of hydrogen (Schneider and Schlegel,Biochim. Biophys. Acta 452, 66-80 (1976); Burgdorf, J. Bact. 187(9)3122-3132(2005)). Soluble EM16 enzymes are additionally present inseveral other organisms including Geobacter sulfurreducens (Coppi,Microbiology 151, 1239-1254 (2005)), Synechocystis str. PCC 6803(Germer, J. Biol. Chem., 284(52), 36462-36472 (2009)), and Thiocapsaroseopersicina (Rakhely, Appl. Environ. Microbiol. 70(2) 722-728(2004)). The Synechocystis enzyme is capable of generating NADPH fromhydrogen. Overexpression of both the Hox operon from Synechocystis str.PCC 6803 and the accessory genes encoded by the Hyp operon from Nostocsp. PCC 7120 led to increased EM16 activity compared to expression ofthe Hox genes alone (Germer, J. Biol. Chem. 284(52), 36462-36472(2009)).

Protein GenBank ID GI Number Organism HoxF NP_942727.1 38637753Ralstonia eutropha H16 HoxU NP_942728.1 38637754 Ralstonia eutropha H16HoxY NP_942729.1 38637755 Ralstonia eutropha H16 HoxH NP_942730.138637756 Ralstonia eutropha H16 HoxW NP_942731.1 38637757 Ralstoniaeutropha H16 HoxI NP_942732.1 38637758 Ralstonia eutropha H16 HoxENP_953767.1 39997816 Geobacter sulfurreducens HoxF NP_953766.1 39997815Geobacter sulfurreducens HoxU NP_953765.1 39997814 Geobactersulfurreducens HoxY NP_953764.1 39997813 Geobacter sulfurreducens HoxHNP_953763.1 39997812 Geobacter sulfurreducens GSU2717 NP_953762.139997811 Geobacter sulfurreducens HoxE NP_441418.1 16330690Synechocystis str. PCC 6803 HoxF NP_441417.1 16330689 Synechocystis str.PCC 6803 Unknown NP_441416.1 16330688 Synechocystis str. PCC 6803function HoxU NP_441415.1 16330687 Synechocystis str. PCC 6803 HoxYNP_441414.1 16330686 Synechocystis str. PCC 6803 Unknown NP_441413.116330685 Synechocystis str. PCC 6803 function Unknown NP_441412.116330684 Synechocystis str. PCC 6803 function HoxH NP_441411.1 16330683Synechocystis str. PCC 6803 HypF NP_484737.1 17228189 Nostoc sp. PCC7120 HypC NP_484738.1 17228190 Nostoc sp. PCC 7120 HypD NP_484739.117228191 Nostoc sp. PCC 7120 Unknown NP_484740.1 17228192 Nostoc sp. PCC7120 function HypE NP_484741.1 17228193 Nostoc sp. PCC 7120 HypANP_484742.1 17228194 Nostoc sp. PCC 7120 HypB NP_484743.1 17228195Nostoc sp. PCC 7120 Hox1E AAP50519.1 37787351 Thiocapsa roseopersicinaHox1F AAP50520.1 37787352 Thiocapsa roseopersicina Hox1U AAP50521.137787353 Thiocapsa roseopersicina Hox1Y AAP50522.1 37787354 Thiocapsaroseopersicina Hox1H AAP50523.1 37787355 Thiocapsa roseopersicina

The genomes of E. coli and other enteric bacteria encode up to four EM16enzymes (Sawers, G., Antonie Van Leeuwenhoek 66:57-88 (1994); Sawers etal., J Bacteriol. 164:1324-1331 (1985); Sawers and Boxer, Eur. JBiochem. 156:265-275 (1986); Sawers et al., J Bacteriol. 168:398-404(1986)). Given the multiplicity of enzyme activities E. coli or anotherhost organism can provide sufficient EM16 activity to split incomingmolecular hydrogen and reduce the corresponding acceptor. Endogenoushydrogen-lyase enzymes of E. coli include hydrogenase 3, amembrane-bound enzyme complex using ferredoxin as an acceptor, andhydrogenase 4 that also uses a ferredoxin acceptor. Hydrogenase 3 and 4are encoded by the hyc and hyf gene clusters, respectively. EM16activity in E. coli is also dependent upon the expression of the hypgenes whose corresponding proteins are involved in the assembly of theEM16 complexes (Jacobi et al., Arch. Microbiol 158:444-451 (1992);Rangarajan et al., J Bacteriol. 190:1447-1458 (2008)). The M.thermoacetica and Clostridium ljungdahli EM16s are suitable for a hostthat lacks sufficient endogenous EM16 activity. M. thermoacetica and C.ljungdahli can grow with CO₂ as the exclusive carbon source indicatingthat reducing equivalents are extracted from H₂ to enable acetyl-CoAsynthesis via the Wood-Ljungdahl pathway (Drake, H. L., J Bacteriol.150:702-709 (1982); Drake and Daniel, Res Microbiol 155:869-883 (2004);Kellum and Drake, J Bacteriol. 160:466-469 (1984)). M. thermoacetica hashomologs to several hyp, hyc, and hyf genes from E. coli. These proteinsequences encoded for by these genes are identified by the followingGenBank accession numbers. In addition, several gene clusters encodingEM16 functionality are present in M. thermoacetica and C. ljungdahli(see for example US 2012/0003652).

Protein GenBank ID GI Number Organism HypA NP_417206 16130633Escherichia coli HypB NP_417207 16130634 Escherichia coli HypC NP_41720816130635 Escherichia coli HypD NP_417209 16130636 Escherichia coli HypENP_417210 226524740 Escherichia coli HypF NP_417192 16130619 Escherichiacoli HycA NP_417205 16130632 Escherichia coli HycB NP_417204 16130631Escherichia coli HycC NP_417203 16130630 Escherichia coli HycD NP_41720216130629 Escherichia coli HycE NP_417201 16130628 Escherichia coli HycFNP_417200 16130627 Escherichia coli HycG NP_417199 16130626 Escherichiacoli HycH NP_417198 16130625 Escherichia coli HycI NP_417197 16130624Escherichia coli HyfA NP_416976 90111444 Escherichia coli HyfB NP_41697716130407 Escherichia coli HyfC NP_416978 90111445 Escherichia coli HyfDNP_416979 16130409 Escherichia coli HyfE NP_416980 16130410 Escherichiacoli HyfF NP_416981 16130411 Escherichia coli HyfG NP_416982 16130412Escherichia coli HyfH NP_416983 16130413 Escherichia coli HyfI NP_41698416130414 Escherichia coli HyfJ NP_416985 90111446 Escherichia coli HyfRNP_416986 90111447 Escherichia coli

Proteins in M. thermoacetica whose genes are homologous to the E. coliEM16 genes are shown below.

Protein GenBank ID GI Number Organism Moth_2175 YP_431007 83590998Moorella thermoacetica Moth_2176 YP_431008 83590999 Moorellathermoacetica Moth_2177 YP_431009 83591000 Moorella thermoaceticaMoth_2178 YP_431010 83591001 Moorella thermoacetica Moth_2179 YP_43101183591002 Moorella thermoacetica Moth_2180 YP_431012 83591003 Moorellathermoacetica Moth_2181 YP_431013 83591004 Moorella thermoaceticaMoth_2182 YP_431014 83591005 Moorella thermoacetica Moth_2183 YP_43101583591006 Moorella thermoacetica Moth_2184 YP_431016 83591007 Moorellathermoacetica Moth_2185 YP_431017 83591008 Moorella thermoaceticaMoth_2186 YP_431018 83591009 Moorella thermoacetica Moth_2187 YP_43101983591010 Moorella thermoacetica Moth_2188 YP_431020 83591011 Moorellathermoacetica Moth_2189 YP_431021 83591012 Moorella thermoaceticaMoth_2190 YP_431022 83591013 Moorella thermoacetica Moth_2191 YP_43102383591014 Moorella thermoacetica Moth_2192 YP_431024 83591015 Moorellathermoacetica Moth_0439 YP_429313 83589304 Moorella thermoaceticaMoth_0440 YP_429314 83589305 Moorella thermoacetica Moth_0441 YP_42931583589306 Moorella thermoacetica Moth_0442 YP_429316 83589307 Moorellathermoacetica Moth_0809 YP_429670 83589661 Moorella thermoaceticaMoth_0810 YP_429671 83589662 Moorella thermoacetica Moth_0811 YP_42967283589663 Moorella thermoacetica Moth_0812 YP_429673 83589664 Moorellathermoacetica Moth_0814 YP_429674 83589665 Moorella thermoaceticaMoth_0815 YP_429675 83589666 Moorella thermoacetica Moth_0816 YP_42967683589667 Moorella thermoacetica Moth_1193 YP_430050 83590041 Moorellathermoacetica Moth_1194 YP_430051 83590042 Moorella thermoaceticaMoth_1195 YP_430052 83590043 Moorella thermoacetica Moth_1196 YP_43005383590044 Moorella thermoacetica Moth_1717 YP_430562 83590553 Moorellathermoacetica Moth_1718 YP_430563 83590554 Moorella thermoaceticaMoth_1719 YP_430564 83590555 Moorella thermoacetica Moth_1883 YP_43072683590717 Moorella thermoacetica Moth_1884 YP_430727 83590718 Moorellathermoacetica Moth_1885 YP_430728 83590719 Moorella thermoaceticaMoth_1886 YP_430729 83590720 Moorella thermoacetica Moth_1887 YP_43073083590721 Moorella thermoacetica Moth_1888 YP_430731 83590722 Moorellathermoacetica Moth_1452 YP_430305 83590296 Moorella thermoaceticaMoth_1453 YP_430306 83590297 Moorella thermoacetica Moth_1454 YP_43030783590298 Moorella thermoacetica

Genes encoding EM16 enzymes from C. ljungdahli are shown below.

Protein GenBank ID GI Number Organism CLJU_c20290 ADK15091.1 300435324Clostridium ljungdahli CLJU_c07030 ADK13773.1 300434006 Clostridiumljungdahli CLJU_c07040 ADK13774.1 300434007 Clostridium ljungdahliCLJU_c07050 ADK13775.1 300434008 Clostridium ljungdahli CLJU_c07060ADK13776.1 300434009 Clostridium ljungdahli CLJU_c07070 ADK13777.1300434010 Clostridium ljungdahli CLJU_c07080 ADK13778.1 300434011Clostridium ljungdahli CLJU_c14730 ADK14541.1 300434774 Clostridiumljungdahli CLJU_c14720 ADK14540.1 300434773 Clostridium ljungdahliCLJU_c14710 ADK14539.1 300434772 Clostridium ljungdahli CLJU_c14700ADK14538.1 300434771 Clostridium ljungdahli CLJU_c28670 ADK15915.1300436148 Clostridium ljungdahli CLJU_c28660 ADK15914.1 300436147Clostridium ljungdahli CLJU_c28650 ADK15913.1 300436146 Clostridiumljungdahli CLJU_c28640 ADK15912.1 300436145 Clostridium ljungdahli

In some cases, EM16 encoding genes are located adjacent to a CODH. InRhodospirillum rubrum, the encoded CODH/hydrogenase proteins form amembrane-bound enzyme complex that has been indicated to be a site whereenergy, in the form of a proton gradient, is generated from theconversion of CO and H₂O to CO₂ and H₂ (Fox et al., J Bacteriol.178:6200-6208 (1996)). The CODH-I of C. hydrogenoformans and itsadjacent genes have been proposed to catalyze a similar functional rolebased on their similarity to the R. rubrum CODH/hydrogenase gene cluster(Wu et al., PLoS Genet. 1:e65 (2005)). The C. hydrogenoformans CODH-Iwas also shown to exhibit intense CO oxidation and CO₂ reductionactivities when linked to an electrode (Parkin et al., J Am. Chem. Soc.129:10328-10329 (2007)).

Protein GenBank ID GI Number Organism CooL AAC45118 1515468Rhodospirillum rubrum CooX AAC45119 1515469 Rhodospirillum rubrum CooUAAC45120 1515470 Rhodospirillum rubrum CooH AAC45121 1498746Rhodospirillum rubrum CooF AAC45122 1498747 Rhodospirillum rubrum CODH(CooS) AAC45123 1498748 Rhodospirillum rubrum CooC AAC45124 1498749Rhodospirillum rubrum CooT AAC45125 1498750 Rhodospirillum rubrum CooJAAC45126 1498751 Rhodospirillum rubrum CODH-I (CooS-I) YP_36064478043418 Carboxydothermus hydrogenoformans CooF YP_360645 78044791Carboxydothermus hydrogenoformans HypA YP_360646 78044340Carboxydothermus hydrogenoformans CooH YP_360647 78043871Carboxydothermus hydrogenoformans CooU YP_360648 78044023Carboxydothermus hydrogenoformans CooX YP_360649 78043124Carboxydothermus hydrogenoformans CooL YP_360650 78043938Carboxydothermus hydrogenoformans CooK YP_360651 78044700Carboxydothermus hydrogenoformans CooM YP_360652 78043942Carboxydothermus hydrogenoformans CooC YP_360654.1 78043296Carboxydothermus hydrogenoformans CooA-1 YP_360655.1 78044021Carboxydothermus_hydrogenoformans

Some EM16 and CODH enzymes transfer electrons to ferredoxins.Ferredoxins are small acidic proteins containing one or more iron-sulfurclusters that function as intracellular electron carriers with a lowreduction potential. Reduced ferredoxins donate electrons toFe-dependent enzymes such as ferredoxin-NADP⁺ oxidoreductase,pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxinoxidoreductase (OFOR). The H. thermophilus gene fdx1 encodes a[4Fe-4S]-type ferredoxin that is required for the reversiblecarboxylation of 2-oxoglutarate and pyruvate by OFOR and PFOR,respectively (Yamamoto et al., Extremophiles 14:79-85 (2010)). Theferredoxin associated with the Sulfolobus solfataricus2-oxoacid:ferredoxin reductase is a monomeric dicluster [3Fe-4S][4Fe-4S]type ferredoxin (Park et al. 2006). While the gene associated with thisprotein has not been fully sequenced, the N-terminal domain shares 93%homology with the zfx ferredoxin from S. acidocaldarius. The E. coligenome encodes a soluble ferredoxin of unknown physiological function,fdx. Some evidence indicates that this protein can function iniron-sulfur cluster assembly (Takahashi and Nakamura, 1999). Additionalferredoxin proteins have been characterized in Helicobacter pylori(Mukhopadhyay et al. 2003) and Campylobacter jejuni (van Vliet et al.2001). A 2Fe-2S ferredoxin from Clostridium pasteurianum has been clonedand expressed in E. coli (Fujinaga and Meyer, Biochemical andBiophysical Research Communications, 192(3): (1993)). Acetogenicbacteria such as Moorella thermoacetica, Clostridium carboxidivorans P7,Clostridium ljungdahli and Rhodospirillum rubrum are predicted to encodeseveral ferredoxins, listed below.

Protein GenBank ID GI Number Organism fdx1 BAE02673.1 68163284Hydrogenobacter thermophilus M11214.1 AAA83524.1 144806 Clostridiumpasteurianum Zfx AAY79867.1 68566938 Sulfolobus acidocalarius FdxAAC75578.1 1788874 Escherichia coli hp_0277 AAD07340.1 2313367Helicobacter pylori fdxA CAL34484.1 112359698 Campylobacter jejuniMoth_0061 ABC18400.1 83571848 Moorella thermoacetica Moth_1200ABC19514.1 83572962 Moorella thermoacetica Moth_1888 ABC20188.1 83573636Moorella thermoacetica Moth_2112 ABC20404.1 83573852 Moorellathermoacetica Moth_1037 ABC19351.1 83572799 Moorella thermoaceticaCcarbDRAFT_4383 ZP_05394383.1 255527515 Clostridium carboxidivorans P7CcarbDRAFT_2958 ZP_05392958.1 255526034 Clostridium carboxidivorans P7CcarbDRAFT_2281 ZP_05392281.1 255525342 Clostridium carboxidivorans P7CcarbDRAFT_5296 ZP_05395295.1 255528511 Clostridium carboxidivorans P7CcarbDRAFT_1615 ZP_05391615.1 255524662 Clostridium carboxidivorans P7CcarbDRAFT_1304 ZP_05391304.1 255524347 Clostridium carboxidivorans P7cooF AAG29808.1 11095245 Carboxydothermus hydrogenoformans fdxNCAA35699.1 46143 Rhodobacter capsulatus Rru_A2264 ABC23064.1 83576513Rhodospirillum rubrum Rru_A1916 ABC22716.1 83576165 Rhodospirillumrubrum Rru_A2026 ABC22826.1 83576275 Rhodospirillum rubrum cooFAAC45122.1 1498747 Rhodospirillum rubrum fdxN AAA26460.1 152605Rhodospirillum rubrum Alvin_2884 ADC63789.1 288897953 Allochromatiumvinosum DSM 180 Fdx YP_002801146.1 226946073 Azotobacter vinelandii DJCKL_3790 YP_001397146.1 153956381 Clostridium kluyveri DSM 555 fer1NP_949965.1 39937689 Rhodopseudomonas palustris CGA009 Fdx CAA12251.13724172 Thauera aromatica CHY_2405 YP_361202.1 78044690 Carboxydothermushydrogenoformans Fer YP_359966.1 78045103 Carboxydothermushydrogenoformans Fer AAC83945.1 1146198 Bacillus subtilis fdx1NP_249053.1 15595559 Pseudomonas aeruginosa PA01 yfhL AP_003148.189109368 Escherichia coli K-12 CLJU_c00930 ADK13195.1 300433428Clostridium ljungdahli CLJU_c00010 ADK13115.1 300433348 Clostridiumljungdahli CLJU_c01820 ADK13272.1 300433505 Clostridium ljungdahliCLJU_c17980 ADK14861.1 300435094 Clostridium ljungdahli CLJU_c17970ADK14860.1 300435093 Clostridium ljungdahli CLJU_c22510 ADK15311.1300435544 Clostridium ljungdahli CLJU_c26680 ADK15726.1 300435959Clostridium ljungdahli CLJU_c29400 ADK15988.1 300436221 Clostridiumljungdahli

Ferredoxin oxidoreductase enzymes transfer electrons from ferredoxins orflavodoxins to NAD(P)H. Two enzymes catalyzing the reversible transferof electrons from reduced ferredoxins to NAD(P)+ are ferredoxin:NAD+oxidoreductase (EC 1.18.1.3) and ferredoxin:NADP+ oxidoreductase (FNR,EC 1.18.1.2). Ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) has anoncovalently bound FAD cofactor that facilitates the reversibletransfer of electrons from NADPH to low-potential acceptors such asferredoxins or flavodoxins (Blaschkowski et al., Eur. J. Biochem.123:563-569 (1982); Fujii et al., 1977). The Helicobacter pylori FNR,encoded by HP1164 (fqrB), is coupled to the activity ofpyruvate:ferredoxin oxidoreductase (PFOR) resulting in thepyruvate-dependent production of NADPH (St et al. 2007). An analogousenzyme is found in Campylobacter jejuni (St Maurice et al., J.Bacteriol. 189:4764-4773 (2007)). A ferredoxin:NADP+ oxidoreductaseenzyme is encoded in the E. coli genome by fpr (Bianchi et al. 1993).Ferredoxin:NAD+ oxidoreductase utilizes reduced ferredoxin to generateNADH from NAD+. In several organisms, including E. coli, this enzyme isa component of multifunctional dioxygenase enzyme complexes. Theferredoxin:NAD+ oxidoreductase of E. coli, encoded by hcaD, is acomponent of the 3-phenylproppionate dioxygenase system involved ininvolved in aromatic acid utilization (Diaz et al. 1998).NADH:ferredoxin reductase activity was detected in cell extracts ofHydrogenobacter thermophilus, although a gene with this activity has notyet been indicated (Yoon et al. 2006). Additional ferredoxin:NAD(P)+oxidoreductases have been annotated in Clostridium carboxydivorans P7.The NADH-dependent reduced ferredoxin: NADP oxidoreductase of C.kluyveri, encoded by nfnAB, catalyzes the concomitant reduction offerredoxin and NAD+ with two equivalents of NADPH (Wang et al, JBacteriol 192: 5115-5123 (2010)). Finally, the energy-conservingmembrane-associated Rnf-type proteins (Seedorf et al, PNAS 105:2128-2133(2008); and Herrmann, J. Bacteriol 190:784-791 (2008)) provide a meansto generate NADH or NADPH from reduced ferredoxin.

Protein GenBank ID GI Number Organism fqrB NP_207955.1 15645778Helicobacter pylori fqrB YP_001482096.1 157414840 Campylobacter jejuniRPA3954 CAE29395.1 39650872 Rhodopseudomonas palustris Fpr BAH29712.1225320633 Hydrogenobacter thermophilus yumC NP_391091.2 255767736Bacillus subtilis Fpr P28861.4 399486 Escherichia coli hcaD AAC75595.11788892 Escherichia coli LOC100282643 NP_001149023.1 226497434 Zea maysNfnA YP_001393861.1 153953096 Clostridium kluyveri NfnB YP_001393862.1153953097 Clostridium kluyveri CcarbDRAFT_2639 ZP_05392639.1 255525707Clostridium carboxidivorans P7 CcarbDRAFT_2638 ZP_05392638.1 255525706Clostridium carboxidivorans P7 CcarbDRAFT_2636 ZP_05392636.1 255525704Clostridium carboxidivorans P7 CcarbDRAFT_5060 ZP_05395060.1 255528241Clostridium carboxidivorans P7 CcarbDRAFT_2450 ZP_05392450.1 255525514Clostridium carboxidivorans P7 CcarbDRAFT_1084 ZP_05391084.1 255524124Clostridium carboxidivorans P7 RnfC EDK33306.1 146346770 Clostridiumkluyveri RnfD EDK33307.1 146346771 Clostridium kluyveri RnfG EDK33308.1146346772 Clostridium kluyveri RnfE EDK33309.1 146346773 Clostridiumkluyveri RnfA EDK33310.1 146346774 Clostridium kluyveri RnfB EDK33311.1146346775 Clostridium kluyveri CLJU_c11410 (RnfB) ADK14209.1 300434442Clostridium ljungdahlii CLJU_c11400 (RnfA) ADK14208.1 300434441Clostridium ljungdahlii CLJU_c11390 (RnfE) ADK14207.1 300434440Clostridium ljungdahlii CLJU_c11380 (RnfG) ADK14206.1 300434439Clostridium ljungdahlii CLJU_c11370 (RnfD) ADK14205.1 300434438Clostridium ljungdahlii CLJU_c11360 (RnfC) ADK14204.1 300434437Clostridium ljungdahlii MOTH_1518 (NfnA) YP_430370.1 83590361 Moorellathermoacetica MOTH_1517(NfnB) YP_430369.1 83590360 Moorellathermoacetica CHY_1992 (NfnA) YP_360811.1 78045020 Carboxydothermushydrogenoformans CHY_1993 (NfnB) YP_360812.1 78044266 Carboxydothermushydrogenoformans CLJU_c37220 (NfnAB) YP_003781850.1 300856866Clostridium ljungdahliiFIG. 3, Step I—Formate Dehydrogenase (EM8)

Formate dehydrogenase (FDH; EM8) catalyzes the reversible transfer ofelectrons from formate to an acceptor. Enzymes with FDH activity utilizevarious electron carriers such as, for example, NADH (EC 1.2.1.2), NADPH(EC 1.2.1.43), quinols (EC 1.1.5.6), cytochromes (EC 1.2.2.3) and EM16s(EC 1.1.99.33). FDH enzymes have been characterized from Moorellathermoacetica (Andreesen and Ljungdahl, J Bacteriol 116:867-873 (1973);Li et al., J Bacteriol 92:405-412 (1966); Yamamoto et al., J Biol Chem.258:1826-1832 (1983). The loci, Moth_2312 is responsible for encodingthe alpha subunit of EM8 while the beta subunit is encoded by Moth_2314(Pierce et al., Environ Microbiol (2008)). Another set of genes encodingEM8 activity with a propensity for CO₂ reduction is encoded by Sfum_2703through Sfum_2706 in Syntrophobacter fumaroxidans (de Bok et al., Eur JBiochem. 270:2476-2485 (2003)); Reda et al., PNAS 105:10654-10658(2008)). A similar set of genes presumed to carry out the same functionare encoded by CHY_0731, CHY_0732, and CHY_0733 in C. hydrogenoformans(Wu et al., PLoS Genet 1:e65 (2005)). EM8s are also found manyadditional organisms including C. carboxidivorans P7, Bacillusmethanolicus, Burkholderia stabilis, Moorella thermoacetica ATCC 39073,Candida boidinii, Candida methylica, and Saccharomyces cerevisiae S288c.The soluble EM8 from Ralstonia eutropha reduces NAD⁺ (fdsG, -B, -A, -C,-D) (Oh and Bowien, 1998).

Several EM8 enzymes have been identified that have higher specificityfor NADP as the cofactor as compared to NAD. This enzyme has been deemedas the NADP-dependent formate dehydrogenase and has been reported from 5species of the Burkholderia cepacia complex. It was tested and verifiedin multiple strains of Burkholderia multivorans, Burkholderia stabilis,Burkholderia pyrrocinia, and Burkholderia cenocepacia (Hatrongjit etal., Enzyme and Microbial Tech., 46: 557-561 (2010)). The enzyme fromBurkholderia stabilis has been characterized and the apparent K_(m) ofthe enzyme were reported to be 55.5 mM, 0.16 mM and 1.43 mM for formate,NADP, and NAD respectively. More gene candidates can be identified usingsequence homology of proteins deposited in Public databases such asNCBI, JGI and the metagenomic databases.

Protein GenBank ID GI Number Organism Moth_2312 YP_431142 148283121Moorella thermoacetica Moth_2314 YP_431144 83591135 Moorellathermoacetica Sfum_2703 YP_846816.1 116750129 Syntrophobacterfumaroxidans Sfum_2704 YP_846817.1 116750130 Syntrophobacterfumaroxidans Sfum_2705 YP_846818.1 116750131 Syntrophobacterfumaroxidans Sfum_2706 YP_846819.1 116750132 Syntrophobacterfumaroxidans CHY_0731 YP_359585.1 78044572 Carboxydothermushydrogenoformans CHY_0732 YP_359586.1 78044500 Carboxydothermushydrogenoformans CHY_0733 YP_359587.1 78044647 Carboxydothermushydrogenoformans CcarbDRAFT_0901 ZP_05390901.1 255523938 Clostridiumcarboxidivorans P7 CcarbDRAFT_4380 ZP_05394380.1 255527512 Clostridiumcarboxidivorans P7 fdhA, MGA3_06625 EIJ82879.1 387590560 Bacillusmethanolicus MGA3 fdhA, PB1_11719 ZP_10131761.1 387929084 Bacillusmethanolicus PB1 fdhD, MGA3_06630 EIJ82880.1 387590561 Bacillusmethanolicus MGA3 fdhD, PB1_11724 ZP_10131762.1 387929085 Bacillusmethanolicus PB1 fdh ACF35003.1 194220249 Burkholderia stabilis fdhACF35004.1 194220251 Burkholderia pyrrocinia fdh ACF35002.1 194220247Burkholderia cenocepacia fdh ACF35001.1 194220245 Burkholderiamultivorans fdh ACF35000.1 194220243 Burkholderia cepacia FDH1AAC49766.1 2276465 Candida boidinii fdh CAA57036.1 1181204 Candidamethylica FDH2 P0CF35.1 294956522 Saccharomyces cerevisiae S288c FDH1NP_015033.1 6324964 Saccharomyces cerevisiae S288c fdsG YP_725156.1113866667 Ralstonia eutropha fdsB YP_725157.1 113866668 Ralstoniaeutropha fdsA YP_725158.1 113866669 Ralstonia eutropha fdsC YP_725159.1113866670 Ralstonia eutropha fdsD YP_725160.1 113866671 RalstoniaeutrophaFIG. 3, Step J—Methanol Dehydrogenase (EM9)

NAD+ dependent EM9 enzymes (EC 1.1.1.244) catalyze the conversion ofmethanol and NAD+ to formaldehyde and NADH. See the present invention asdescribed herein.

FIG. 3, Step L—Formaldehyde Dehydrogenase (EM11)

Oxidation of formaldehyde to formate is catalyzed by EM11. A NAD+dependent EM11 enzyme is encoded by fdhA of Pseudomonas putida (Ito etal, J Bacteriol 176: 2483-2491 (1994)). Additional EM11 enzymes includethe NAD+ and glutathione independent EM11 from Hyphomicrobium zavarzinii(Jerome et al, Appl Microbiol Biotechnol 77:779-88 (2007)), theglutathione dependent EM11 of Pichia pastoris (Sunga et al, Gene330:39-47 (2004)) and the NAD(P)+ dependent EM11 of Methylobactermarinus (Speer et al, FEMS Microbiol Lett, 121(3):349-55 (1994)).

Protein GenBank ID GI Number Organism fdhA P46154.3 1169603 Pseudomonasputida faoA CAC85637.1 19912992 Hyphomicrobium zavarzinii Fld1CCA39112.1 328352714 Pichia pastoris fdh P47734.2 221222447Methylobacter marinus

In addition to the EM11 enzymes listed above, alternate enzymes andpathways for converting formaldehyde to formate are known in the art.For example, many organisms employ glutathione-dependent formaldehydeoxidation pathways, in which formaldehyde is converted to formate inthree steps via the intermediates S-hydroxymethylglutathione andS-formylglutathione (Vorholt et al, J Bacteriol 182:6645-50 (2000)). Theenzymes of this pathway are EM12 (EC 4.4.1.22), EM13 (EC 1.1.1.284) andEM14 (EC 3.1.2.12).

FIG. 3, Step M—Spontaneous or S-(Hydroxymethyl)Glutathione Synthase(EM12)

While conversion of formaldehyde to S-hydroxymethylglutathione can occurspontaneously in the presence of glutathione, it has been shown byGoenrich et al (Goenrich, et al., J Biol Chem 277(5); 3069-72 (2002))that an enzyme from Paracoccus denitrificans can accelerate thisspontaneous condensation reaction. The enzyme catalyzing the conversionof formaldehyde and glutathione was purified and namedglutathione-dependent formaldehyde-activating enzyme (Gfa). The geneencoding it, which was named gfa, is located directly upstream of thegene for EM13, which catalyzes the subsequent oxidation ofS-hydroxymethylglutathione. Putative proteins with sequence identity toGfa from P. denitrificans are present also in Rhodobacter sphaeroides,Sinorhizobium meliloti, and Mesorhizobium loti.

Protein GenBank ID GI Number Organism Gfa Q51669.3 38257308 Paracoccusdenitrificans Gfa ABP71667.1 145557054 Rhodobacter sphaeroides ATCCI17025 Gfa Q92WX6.1 38257348 Sinorhizobium meliloti 1021 Gfa Q98LU4.238257349 Mesorhizobium loti MAFF303099FIG. 3, Step N—Glutathione-Dependent Formaldehyde Dehydrogenase (EM13)

EM13 (GS-FDH) belongs to the family of class III alcohol dehydrogenases.Glutathione and formaldehyde combine non-enzymatically to formhydroxymethylglutathione, the true substrate of the GS-FDH catalyzedreaction. The product, S-formylglutathione, is further metabolized toformic acid.

Protein GenBank ID GI Number Organism frmA YP_488650.1 388476464Escherichia coli K-12 MG1655 SFA1 NP_010113.1 6320033 Saccharomycescerevisiae S288c flhA AAC44551.1 1002865 Paracoccus denitrificans adhIAAB09774.1 986949 Rhodobacter sphaeroidesFIG. 3, Step O—S-Formylglutathione Hydrolase (EM14)

EM14 is a glutathione thiol esterase found in bacteria, plants andanimals. It catalyzes conversion of S-formylglutathione to formate andglutathione. The fghA gene of P. denitrificans is located in the sameoperon with gfa and flhA, two genes involved in the oxidation offormaldehyde to formate in this organism. In E. coli, FrmB is encoded inan operon with FrmR and FrmA, which are proteins involved in theoxidation of formaldehyde. YeiG of E. coli is a promiscuous serinehydrolase; its highest specific activity is with the substrateS-formylglutathione.

Protein GenBank ID GI Number Organism frmB NP_414889.1 16128340Escherichia coli K-12 MG1655 yeiG AAC75215.1 1788477 Escherichia coliK-12 MG1655 fghA AAC44554.1 1002868 Paracoccus denitrificansExemplary enzymes for the methods of using formaldehyde produced fromthe oxidation of methanol in the formation of intermediates of centralmetabolic pathways for the formation of target product or biomass arefurther described, particularly with respect to FIGS. 3B and 3C.

Provided herein are exemplary pathways, which utilize formaldehydeproduced from the oxidation of methanol (see, e.g., FIG. 3, step J) inthe formation of intermediates of certain central metabolic pathwaysthat can be used for the formation of biomass. Exemplary MMPs forenhancing the availability of reducing equivalents, as well as theproducing formaldehyde from methanol (step J), are provided in FIG. 3.

One exemplary pathway that can utilize formaldehyde produced from theoxidation of methanol (e.g., as provided in FIG. 3) is shown in FIG. 3B,which involves condensation of formaldehyde and D-ribulose-5-phosphateto form H6P by EF1 (FIG. 3B, step A). The enzyme can use Mg²⁺ or Mn²⁺for maximal activity, although other metal ions are useful, and evennon-metal-ion-dependent mechanisms are contemplated. H6P is convertedinto F6P by EF2 (FIG. 3B, step B).

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 3) is shown in FIG. 3C and proceeds throughDHA. EF3 is a special transketolase that first transfers a glycoaldehydegroup from xylulose-5-phosphate to formaldehyde, resulting in theformation of DHA and G3P, which is an intermediate in glycolysis (FIG.3C, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 3C, step B).DHAP can be assimilated into glycolysis and several other pathways.

FIG. 3B, Steps A and B—Hexulose-6-Phosphate Synthase (EF1) (Step A) and6-Phospho-3-Hexuloisomerase (EF2) (Step B)

Both of the EF1 and EF2 enzymes are found in several organisms,including methanotrops and methylotrophs where they have been purified(Kato et al., 2006, BioSci Biotechnol Biochem. 70(1):10-21. In addition,these enzymes have been reported in heterotrophs such as Bacillussubtilis also where they are reported to be involved in formaldehydedetoxification (Mitsui et al., 2003, AEM 69(10):6128-32, Yasueda et al.,1999. J Bac 181(23):7154-60. Genes for these two enzymes from themethylotrophic bacterium Mycobacterium gastri MB19 have been fused andE. coli strains harboring the hps-phi construct showed more efficientutilization of formaldehyde (Orita et al., 2007, Appl MicrobiolBiotechnol. 76:439-445). In some organisms, these two enzymes naturallyexist as a fused version that is bifunctional.

Exemplary candidate genes for H6P synthase are:

Protein GenBank ID GI number Organism Hps AAR39392.1 40074227 Bacillusmethanolicus MGA3 Hps EIJ81375.1 387589055 Bacillus methanolicus PB1RmpA BAA83096.1 5706381 Methylomonas aminofaciens RmpA BAA90546.16899861 Mycobacterium gastri YckG BAA08980.1 1805418 Bacillus subtilis

Exemplary gene candidates for EF2 are:

Protein GenBank ID GI number Organism Phi AAR39393.1 40074228 Bacillusmethanolicus MGA3 Phi EIJ81376.1 387589056 Bacillus methanolicus PB1 PhiBAA83098.1 5706383 Methylomonas aminofaciens RmpB BAA90545.1 6899860Mycobacterium gastri

Candidates for enzymes where both of these functions have been fusedinto a single open reading frame include the following.

Protein GenBank ID GI number Organism PH1938 NP_143767.1 14591680Pyrococcus horikoshii OT3 PF0220 NP_577949.1 18976592 Pyrococcusfuriosus TK0475 YP_182888.1 57640410 Thermococcus kodakaraensisNP_127388.1 14521911 Pyrococcus abyssi MCA2738 YP_115138.1 53803128Methylococcus capsulatasFIG. 3C, Step A—Dihydroxyacetone Synthase (EF3)

Another exemplary pathway that involves the detoxification andassimilation of formaldehyde produced from the oxidation of methanol(e.g., as provided in FIG. 3) is shown in FIG. 3C and proceeds throughDHA. EF3 is a special transketolase that first transfers a glycoaldehydegroup from xylulose-5-phosphate to formaldehyde, resulting in theformation of DHA and G3P, which is an intermediate in glycolysis (FIG.3C, step A). The DHA obtained from DHA synthase is then furtherphosphorylated to form DHA phosphate by a DHA kinase (FIG. 3C, step B).DHAP can be assimilated into glycolysis and several other pathways.

The EF3 enzyme in Candida boidinii uses thiamine pyrophosphate and Mg²⁺as cofactors and is localized in the peroxisome. The enzyme from themethanol-growing carboxydobacterium, Mycobacter sp. strain JC1 DSM 3803,was also found to have DHA synthase and kinase activities (Ro et al.,1997, J Bac 179(19):6041-7). DHA synthase from this organism also hassimilar cofactor requirements as the enzyme from C. boidinii. The K_(m)sfor formaldehyde and xylulose 5-phosphate were reported to be 1.86 mMand 33.3 microM, respectively. Several other mycobacteria, excludingonly Mycobacterium tuberculosis, can use methanol as the sole source ofcarbon and energy and are reported to use EF3 (Part et al., 2003, J Bac185(1):142-7.

Protein GenBank ID GI number Organism DAS1 AAC83349.1 3978466 Candidaboidinii HPODL_2613 EFW95760.1 320581540 Ogataea parapolymorpha DL-1(Hansenula polymorpha DL-1) AAG12171.2 18497328 Mycobacter sp. strainJC1 DSM 3803FIG. 3C, Step B—Dihydroxyacetone (DHA) Kinase

DHA obtained from DHA synthase is further phosphorylated to form DHAphosphate by a DHA kinase. DHAP can be assimilated into glycolysis andseveral other pathways. EF4 has been purified from Ogataea angusta tohomogeneity (Bystrkh, 1983, Biokhimiia, 48(10):1611-6). The enzyme,which phosphorylates DHA and, to a lesser degree, glyceraldehyde, is ahomodimeric protein of 139 kDa. ATP is the preferred phosphate groupdonor for the enzyme. When ITP, GTP, CTP and UTP are used, the activitydrops to about 30%. In several organisms such as Klebsiella pneumoniaeand Citrobacter fruendii (Daniel et al., 1995, J Bac 177(15):4392-40),DHA is formed as a result of oxidation of glycerol and is converted intoDHAP by the kinase DHA kinase of K. pneumoniae has been characterized(Jonathan et al, 1984, J Bac 160(1):55-60). It is very specific for DHA,with a K_(m) of 4 μM, and has two apparent K_(m) values for ATP, one at25 to 35 μM, and the other at 200 to 300 μM. DHA can also bephosphorylated by glycerol kinases but the DHA kinase from K. puemoniaeis different from glycerol kinase in several respects. While bothenzymes can phosphorylate DHA, DHA kinase does not phosphorylateglycerol, neither is it inhibited by fructose-1,6-diphosphate. InSaccharomyces cerevisiae, DHA kinases (I and II) are involved inrescuing the cells from toxic effects of DHA (Molin et al., 2003, J BiolChem. 17; 278(3):1415-23).

In Escherichia coli, DHA kinase is composed of the three subunits DhaK,DhaL, and DhaM and it functions similarly to a phosphotransferase system(PTS) in that it utilizes phosphoenolpyruvate as a phosphoryl donor(Gutknecht et al., 2001, EMBO J. 20(10):2480-6). It differs in not beinginvolved in transport. The phosphorylation reaction requires thepresence of the EI and HPr proteins of the PTS system. The DhaM subunitis phosphorylated at multiple sites. DhaK contains the substrate bindingsite (Garcia-Alles et al., 2004, 43(41):13037-45; Siebold et al., 2003,PNAS. 100(14):8188-92). The K_(M) for DHA for the E. coli enzyme hasbeen reported to be 6 μM. The K subunit is similar to the N-terminalhalf of ATP-dependent EF4 of Citrobacter freundii and eukaryotes.

Exemplary DHA kinase gene candidates for this step are:

Protein GenBank ID GI number Organism DAK1 P54838.1 1706391Saccharomyces cerevisiae S288c DAK2 P43550.1 1169289 Saccharomycescerevisiae S288c D186_20916 ZP_16280678.1 421847542 Citrobacter freundiiDAK2 ZP_18488498.1 425085405 Klebsiella pneumoniae DAK AAC27705.13171001 Ogataea angusta DhaK NP_415718.6 162135900 Escherichia coli DhaLNP_415717.1 16129162 Escherichia coli DhaM NP_415716.4 226524708Escherichia coli

Suitable purification and/or assays to test, e.g., for the production ofBDO can be performed using well known methods. Suitable replicates suchas triplicate cultures can be grown for each engineered strain to betested. For example, product and byproduct formation in the engineeredproduction host can be monitored. The final product and intermediates,and other organic compounds, can be analyzed by methods such as HPLC(High Performance Liquid Chromatography), GC-MS (Gas Chromatography-MassSpectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) orother suitable analytical methods using routine procedures well known inthe art. The release of product in the fermentation broth can also betested with the culture supernatant. Byproducts and residual glucose canbe quantified by HPLC using, for example, a refractive index detectorfor glucose and alcohols, and a UV detector for organic acids (Lin etal., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay anddetection methods well known in the art. The individual enzyme orprotein activities from the exogenous DNA sequences can also be assayedusing methods well known in the art.

The BDO or other target molecules may separated from other components inthe culture using a variety of methods well known in the art. Suchseparation methods include, for example, extraction procedures as wellas methods that include continuous liquid-liquid extraction,pervaporation, evaporation, filtration, membrane filtration (includingreverse osmosis, nanofiltration, ultrafiltration, and microfiltration),membrane filtration with diafiltration, membrane separation, reverseosmosis, electrodialysis, distillation, extractive distillation,reactive distillation, azeotropic distillation, crystallization andrecrystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, carbon adsorption, hydrogenation, and ultrafiltration.All of the above methods are well known in the art.

Examples of target molecule isolation processes include distillation for13BDO, 14BDO, butadiene, methyl vinyl carbinol, 3-buten-1-ol,n-propanol, isopropanol, propylene, and crotyl alcohol; crystallizationfor 6ACA (alternatively it can be converted to caprolactam and thenpurified via distillation as a final step), HMDA, adipic acid orderivatives thereof, succinic acid or derivatives thereof, or any ofcrystallization, distillation, or extraction for methacrylic acid orderivatives thereof.

Target molecules such as13BDO, 14BDO, butadiene, methyl vinyl carbinoln-propanol, isopropanol, propylene, crotyl alcohol; 3-buten-1-ol, 6ACA,HMDA, adipic acid or derviaties thereof, succinic acid or derivativesthereof, or methacrylic acid or derivatives thereof are chemicals usedin commercial and industrial applications. In some embodiments, BDOand/or 4-HB are used in various commercial and industrial applications.Non-limiting examples of such applications include production ofplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like. Moreover, BDO and/or 4-HB are also used asa raw material in the production of a wide range of products includingplastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like.

Accordingly, in some embodiments, provided are biobased plastics,elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like, comprising one or more bioderived BDOand/or 4-HB or bioderived BDO and/or 4-HB intermediate thereof producedby an organism provided herein or produced using a method disclosedherein.

As used herein, the term “bioderived” means derived from or synthesizedby a biological organism and can be considered a renewable resourcesince it can be generated by a biological organism. Such a biologicalorganism, in particular the microbial organisms disclosed herein, canutilize feedstock or biomass, such as, sugars or carbohydrates obtainedfrom an agricultural, plant, bacterial, or animal source. Alternatively,the biological organism can utilize atmospheric carbon. As used herein,the term “biobased” means a product as described above that is composed,in whole or in part, of a bioderived compound of the disclosure. Abiobased or bioderived product is in contrast to a petroleum derivedproduct, wherein such a product is derived from or synthesized frompetroleum or a petrochemical feedstock.

In some embodiments, the disclosure provides plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like,comprising bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HBintermediate thereof, wherein the bioderived BDO and/or 4-HB orbioderived BDO and/or 4-HB intermediate thereof includes all or part ofthe BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof used in theproduction of plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra™, nylons, and the like. Thus, in some aspects, thedisclosure provides a biobased plastics, elastic fibers, polyurethanes,polyesters, including polyhydroxyalkanoates such as P4HB or co-polymersthereof, PTMEG and polyurethane-polyurea copolymers, referred to asspandex, elastane or Lycra™, nylons, and the like, comprising at least2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98% or 100% bioderived BDO and/or 4-HB or bioderived BDO and/or4-HB intermediate thereof as disclosed herein. Additionally, in someaspects, the disclosure provides a biobased plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like,wherein the BDO and/or 4-HB or BDO and/or 4-HB intermediate thereof usedin its production is a combination of bioderived and petroleum derivedBDO and/or 4-HB or BDO and/or 4-HB intermediate thereof. For example, abiobased plastics, elastic fibers, polyurethanes, polyesters, includingpolyhydroxyalkanoates such as P4HB or co-polymers thereof, PTMEG andpolyurethane-polyurea copolymers, referred to as spandex, elastane orLycra™, nylons, and the like, can be produced using 50% bioderived BDOand/or 4-HB and 50% petroleum derived BDO and/or 4-HB or other desiredratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%,40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derivedprecursors, so long as at least a portion of the product comprises abioderived product produced by the microbial organisms disclosed herein.It is understood that methods for producing plastics, elastic fibers,polyurethanes, polyesters, including polyhydroxyalkanoates such as P4HBor co-polymers thereof, PTMEG and polyurethane-polyurea copolymers,referred to as spandex, elastane or Lycra™, nylons, and the like, usingthe bioderived BDO and/or 4-HB or bioderived BDO and/or 4-HBintermediate thereof of the disclosure are well known in the art.

In one embodiment, the product is a plastic. In one embodiment, theproduct is an elastic fiber. In one embodiment, the product is apolyurethane. In one embodiment, the product is a polyester. In oneembodiment, the product is a polyhydroxyalkanoate. In one embodiment,the product is a poly-4-HB. In one embodiment, the product is aco-polymer of poly-4-HB. In one embodiment, the product is apoly(tetramethylene ether) glycol. In one embodiment, the product is apolyurethane-polyurea copolymer. In one embodiment, the product is aspandex. In one embodiment, the product is an elastane. In oneembodiment, the product is a Lycra™. In one embodiment, the product is anylon.

In some embodiments, provided herein is a culture medium comprisingbioderived BDO. In some embodiments, the bioderived BDO is produced byculturing an organism having a MDH protein and BDOP, as provided herein.In certain embodiments, the bioderived BDO has a carbon-12, carbon-13and carbon-14 isotope ratio that reflects an atmospheric carbon dioxideuptake source. In one embodiment, the culture medium is separated from aorganism having a MDH protein and BDOP.

In other embodiments, provided herein is a bioderived BDO. In someembodiments, the bioderived BDO is produced by culturing an organismhaving a MDH protein and BDOP, as provided herein. n some embodiments,the bioderived BDO has an Fm value of at least 80%, at least 85%, atleast 90%, at least 95% or at least 98%. In certain embodiments, thebioderived BDO is a component of culture medium.

In certain embodiments, provided herein is a composition comprising abioderived BDO provided herein, for example, a bioderived BDO producedby culturing an orgaism having a MDH protein and BDOP, as providedherein. In some embodiments, the composition further comprises acompound other than said bioderived BDO. In certain embodiments, thecompound other than said bioderived BDO is a trace amount of a cellularportion of an organism having a MDH protein and a BDOP, as providedherein.

In some embodiments, provided herein is a biobased product comprising abioderived BDO provided herein. In certain embodiments, the biobasedproduct is a plastic, elastic fiber, polyurethane, polyester,polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB,poly(tetramethylene ether) glycol, polyurethane-polyurea copolymer,spandex, elastane, Lycra™, or nylon. In certain embodiments, thebiobased product comprises at least 5% bioderived BDO. In certainembodiments, the biobased product is (i) a polymer, THF or a THFderivative, or GBL or a GBL derivative; (ii) a plastic, elastic fiber,polyurethane, polyester, polyhydroxyalkanoate, poly-4-HB, co-polymer ofpoly-4-HB, poly(tetramethylene ether) glycol, polyurethane-polyureacopolymer, spandex, elastane, Lycra™, or nylon; (iii) a polymer, aresin, a fiber, a bead, a granule, a pellet, a chip, a plastic, apolyester, a thermoplastic polyester, a molded article, aninjection-molded article, an injection-molded part, an automotive part,an extrusion resin, an electrical part and a casing; and optionallywhere the biobased product is reinforced or filled and further where thebiobased product is glass-reinforced or -filled or mineral-reinforced or-filled; (iv) a polymer, wherein the polymer comprises polybutyleneterephthalate (PBT); (v) a polymer, wherein the polymer comprises PBTand the biobased product is a resin, a fiber, a bead, a granule, apellet, a chip, a plastic, a polyester, a thermoplastic polyester, amolded article, an injection-molded article, an injection-molded part,an automotive part, an extrusion resin, an electrical part and a casing;and optionally where the biobased product is reinforced or filled andfurther where the biobased product is glass-reinforced or -filled ormineral-reinforced or -filled; (vi) a THF or a THF derivative, whereinthe THF derivative is polytetramethylene ether glycol (PTMEG), apolyester ether (COPE) or a thermoplastic polyurethane; (viii) a THFderivative, wherein the THF derivative comprises a fiber; or (ix) a GBLor a GBL derivative, wherein the GBL derivative is a pyrrolidone. Incertain embodiments, the biobased product comprises at least 10%bioderived BDO. In some embodiments, the biobased product comprises atleast 20% bioderived BDO. In other embodiments, the biobased productcomprises at least 30% bioderived BDO. In some embodiments, the biobasedproduct comprises at least 40% bioderived BDO. In other embodiments, thebiobased product comprises at least 50% bioderived BDO. In oneembodiment, the biobased product comprises a portion of said bioderivedBDO as a repeating unit. In another embodiment, provided herein is amolded product obtained by molding the biobased product provided herein.In other embodiments, provided herein is a process for producing abiobased product provided herein, comprising chemically reacting saidbioderived-BDO with itself or another compound in a reaction thatproduces said biobased product. In certain embodiments, provided hereinis a polymer comprising or obtained by converting the bioderived BDO. Inother embodiments, provided herein is a method for producing a polymer,comprising chemically or enzymatically converting the bioderived BDO tothe polymer. In yet other embodiments, provided herein is a compositioncomprising the bioderived BDO, or a cell lysate or culture supernatantthereof.

In some embodiments, the carbon feedstock and other cellular uptakesources such as phosphate, ammonia, sulfate, chloride and other halogenscan be chosen to alter the isotopic distribution of the atoms present inBDO and/or 4-HB or any BDO and/or 4-HB pathway intermediate. The variouscarbon feedstock and other uptake sources enumerated above will bereferred to herein, collectively, as “uptake sources.” Uptake sourcescan provide isotopic enrichment for any atom present in the product BDOand/or 4-HB or BDO and/or 4-HB pathway intermediate, or for sideproducts generated in reactions diverging away from a BDO and/or 4-HBpathway. Isotopic enrichment can be achieved for any target atomincluding, for example, carbon, hydrogen, oxygen, nitrogen, sulfur,phosphorus, chloride or other halogens. The same holds true for the MMPsand FAPs, as well as intermediates thereof, provided herein.

In some embodiments, the uptake sources can be selected to alter thecarbon-12, carbon-13, and carbon-14 ratios. In some embodiments, theuptake sources can be selected to alter the oxygen-16, oxygen-17, andoxygen-18 ratios. In some embodiments, the uptake sources can beselected to alter the hydrogen, deuterium, and tritium ratios. In someembodiments, the uptake sources can selected to alter the nitrogen-14and nitrogen-15 ratios. In some embodiments, the uptake sources can beselected to alter the sulfur-32, sulfur-33, sulfur-34, and sulfur-35ratios. In some embodiments, the uptake sources can be selected to alterthe phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In someembodiments, the uptake sources can be selected to alter thechlorine-35, chlorine-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be variedto a desired ratio by selecting one or more uptake sources. An uptakesource can be derived from a natural source, as found in nature, or froma man-made source, and one skilled in the art can select a naturalsource, a man-made source, or a combination thereof, to achieve adesired isotopic ratio of a target atom. An example of a man-made uptakesource includes, for example, an uptake source that is at leastpartially derived from a chemical synthetic reaction. Such isotopicallyenriched uptake sources can be purchased commercially or prepared in thelaboratory and/or optionally mixed with a natural source of the uptakesource to achieve a desired isotopic ratio. In some embodiments, atarget isotopic ratio of an uptake source can be obtained by selecting adesired origin of the uptake source as found in nature. For example, asdiscussed herein, a natural source can be a biobased derived from orsynthesized by a biological organism or a source such as petroleum-basedproducts or the atmosphere. In some such embodiments, a source ofcarbon, for example, can be selected from a fossil fuel-derived carbonsource, which can be relatively depleted of carbon-14, or anenvironmental or atmospheric carbon source, such as CO₂, which canpossess a larger amount of carbon-14 than its petroleum-derivedcounterpart.

Isotopic enrichment is readily assessed by mass spectrometry usingtechniques known in the art such as Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC) and/or high performance liquid chromatography (HPLC).

The unstable carbon isotope carbon-14 or radiocarbon makes up forroughly 1 in 10¹² carbon atoms in the earth's atmosphere and has ahalf-life of about 5700 years. The stock of carbon is replenished in theupper atmosphere by a nuclear reaction involving cosmic rays andordinary nitrogen (¹⁴N). Fossil fuels contain no carbon-14, as itdecayed long ago. Burning of fossil fuels lowers the atmosphericcarbon-14 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound arewell known to those skilled in the art. Isotopic enrichment is readilyassessed by mass spectrometry using techniques known in the art such asaccelerated mass spectrometry (AMS), Stable Isotope Ratio MassSpectrometry (SIRMS) and Site-Specific Natural Isotopic Fractionation byNuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques canbe integrated with separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC) and/or gaschromatography, and the like.

In the case of carbon, ASTM D6866 was developed in the United States asa standardized analytical method for determining the biobased content ofsolid, liquid, and gaseous samples using radiocarbon dating by theAmerican Society for Testing and Materials (ASTM) International. Thestandard is based on the use of radiocarbon dating for the determinationof a product's biobased content. ASTM D6866 was first published in 2004,and the current active version of the standard is ASTM D6866-11(effective Apr. 1, 2011). Radiocarbon dating techniques are well knownto those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio ofcarbon-14 (¹⁴C) to carbon-12 (¹²C). Specifically, the Fraction Modern(Fm) is computed from the expression: Fm=(S−B)/(M−B), where B, S and Mrepresent the ¹⁴C/¹²C ratios of the blank, the sample and the modernreference, respectively. Fraction Modern is a measurement of thedeviation of the ¹⁴C/¹²C ratio of a sample from “Modern.” Modern isdefined as 95% of the radiocarbon concentration (in AD 1950) of NationalBureau of Standards (NBS) Oxalic Acid I (i.e., standard referencematerials (SRM) 4990b) normalized to δ¹³C_(VPDB)=−19 per mil (Olsson,The use of Oxalic acid as a Standard. in, Radiocarbon Variations andAbsolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, NewYork (1970)). Mass spectrometry results, for example, measured by ASM,are calculated using the internationally agreed upon definition of 0.95times the specific activity of NBS Oxalic Acid I (SRM 4990b) normalizedto δ¹³C_(VPDB)=−19 per mil. This is equivalent to an absolute (AD 1950)¹⁴C/¹²C ratio of 1.176±0.010×10⁻¹² (Karlen et al., Arkiv Geofysik,4:465-471 (1968)). The standard calculations take into account thedifferential uptake of one isotope with respect to another, for example,the preferential uptake in biological systems of C¹² over C¹³ over C¹⁴,and these corrections are reflected as a Fm corrected for δ¹³.

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of1955 sugar beet. Although there were 1000 lbs made, this oxalic acidstandard is no longer commercially available. The Oxalic Acid IIstandard (HOx 2; N.I.S.T designation SRM 4990 C) was made from a crop of1977 French beet molasses. In the early 1980's, a group of 12laboratories measured the ratios of the two standards. The ratio of theactivity of Oxalic acid II to 1 is 1.2933±0.001 (the weighted mean). Theisotopic ratio of HOx II is −17.8 per mille. ASTM D6866-11 suggests useof the available Oxalic Acid II standard SRM 4990 C (Hox2) for themodern standard (see discussion of original vs. currently availableoxalic acid standards in Mann, Radiocarbon, 25(2):519-527 (1983)). AFm=0% represents the entire lack of carbon-14 atoms in a material, thusindicating a fossil (for example, petroleum based) carbon source. AFm=100%, after correction for the post-1950 injection of carbon-14 intothe atmosphere from nuclear bomb testing, indicates an entirely moderncarbon source. As described herein, such a “modern” source includesbiobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can begreater than 100% because of the continuing but diminishing effects ofthe 1950s nuclear testing programs, which resulted in a considerableenrichment of carbon-14 in the atmosphere as described in ASTM D6866-11.Because all sample carbon-14 activities are referenced to a “pre-bomb”standard, and because nearly all new biobased products are produced in apost-bomb environment, all pMC values (after correction for isotopicfraction) must be multiplied by 0.95 (as of 2010) to better reflect thetrue biobased content of the sample. A biobased content that is greaterthan 103% suggests that either an analytical error has occurred, or thatthe source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material'stotal organic content and does not consider the inorganic carbon andother non-carbon containing substances present. For example, a productthat is 50% starch-based material and 50% water would be considered tohave a Biobased Content=100% (50% organic content that is 100% biobased)based on ASTM D6866. In another example, a product that is 50%starch-based material, 25% petroleum-based, and 25% water would have aBiobased Content=66.7% (75% organic content but only 50% of the productis biobased). In another example, a product that is 50% organic carbonand is a petroleum-based product would be considered to have a BiobasedContent=0% (50% organic carbon but from fossil sources). Thus, based onthe well known methods and known standards for determining the biobasedcontent of a compound or material, one skilled in the art can readilydetermine the biobased content and/or prepared downstream productshaving a desired biobased content.

Applications of carbon-14 dating techniques to quantify bio-basedcontent of materials are known in the art (Currie et al., NuclearInstruments and Methods in Physics Research B, 172:281-287 (2000)). Forexample, carbon-14 dating has been used to quantify bio-based content interephthalate-containing materials (Colonna et al., Green Chemistry,13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT)polymers derived from renewable 1,3-propanediol and petroleum-derivedterephthalic acid resulted in Fm values near 30% (i.e., since 3/11 ofthe polymeric carbon derives from renewable 1,3-propanediol and 8/11from the fossil end member terephthalic acid) (Currie et al., supra,2000). In contrast, polybutylene terephthalate polymer derived from bothrenewable BDO and renewable terephthalic acid resulted in bio-basedcontent exceeding 90% (Colonna et al., supra, 2011).

Accordingly, in some embodiments, provided are BDO and/or 4-HB or a BDOand/or 4-HB pathway intermediate thereof that has a carbon-12,carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, alsoreferred to as environmental carbon, uptake source. For example, in someaspects the BDO and/or 4-HB or a BDO and/or 4-HB intermediate thereofcan have an Fm value of at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or as much as 100%. In some such embodiments, the uptakesource is CO₂. In some embodiments, provided is BDO and/or 4-HB or a BDOand/or 4-HB intermediate thereof that has a carbon-12, carbon-13, andcarbon-14 ratio that reflects petroleum-based carbon uptake source. Inthis aspect, the BDO and/or 4-HB or a BDO and/or 4-HB intermediatethereof can have an Fm value of less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, less than 50%, less than 45%, less than 40%,less than 35%, less than 30%, less than 25%, less than 20%, less than15%, less than 10%, less than 5%, less than 2% or less than 1%. In someembodiments, provided is BDO and/or 4-HB or a BDO and/or 4-HBintermediate thereof that has a carbon-12, carbon-13, and carbon-14ratio that is obtained by a combination of an atmospheric carbon uptakesource with a petroleum-based uptake source. Using such a combination ofuptake sources is one way by which the carbon-12, carbon-13, andcarbon-14 ratio can be varied, and the respective ratios would reflectthe proportions of the uptake sources.

Further, the disclosure relates, in part, to biologically produced BDOand/or 4-HB or BDO and/or 4-HB intermediate thereof as disclosed herein,and to the products derived therefrom, wherein the BDO and/or 4-HB or aBDO and/or 4-HB intermediate thereof has a carbon-12, carbon-13, andcarbon-14 isotope ratio of about the same value as the CO₂ that occursin the environment. For example, in some aspects, provided are abioderived BDO and/or 4-HB or a bioderived BDO and/or 4-HB intermediatethereof having a carbon-12 versus carbon-13 versus carbon-14 isotoperatio of about the same value as the CO₂ that occurs in the environment,or any of the other ratios disclosed herein. It is understood, asdisclosed herein, that a product can have a carbon-12 versus carbon-13versus carbon-14 isotope ratio of about the same value as the CO₂ thatoccurs in the environment, or any of the ratios disclosed herein,wherein the product is generated from bioderived BDO and/or 4-HB or abioderived BDO and/or 4-HB intermediate thereof as disclosed herein,wherein the bioderived product is chemically modified to generate afinal product. Methods of chemically modifying a bioderived product ofBDO and/or 4-HB, or an intermediate thereof, to generate a desiredproduct are well known to those skilled in the art, as described herein.Also provided are plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as poly-4-hydroxybutyrate (P4HB) orco-polymers thereof, poly(tetramethylene ether) glycol (PTMEG)(alsoreferred to as PTMO, polytetramethylene oxide) and polyurethane-polyureacopolymers, referred to as spandex, elastane or Lycra™, nylons, and thelike, having a carbon-12 versus carbon-13 versus carbon-14 isotope ratioof about the same value as the CO₂ that occurs in the environment,wherein the plastics, elastic fibers, polyurethanes, polyesters,including polyhydroxyalkanoates such as P4HB or co-polymers thereof,PTMEG and polyurethane-polyurea copolymers, referred to as spandex,elastane or Lycra™, nylons, and the like, are generated directly from orin combination with bioderived BDO and/or 4-HB or a bioderived BDOand/or 4-HB intermediate thereof as disclosed herein.

Those skilled in the art will understand that an organism can beengineered that secretes the biosynthesized compounds when grown on acarbon source such as a methanol alone or combined with othercarbohydrates. Such compounds include, for example, BDO and any of theintermediate metabolites in the BDOP. All that is required is toengineer in one or more of the required enzyme or protein activities toachieve biosynthesis of the desired compound or intermediate including,for example, inclusion of some or all of the BDO biosynthetic pathways.Accordingly, provided herein is an organism that produces and/orsecretes BDO when grown on a carbohydrate or other carbon source andproduces and/or secretes any of the intermediate metabolites shown inthe BDOP when grown on a carbohydrate or other carbon source. The BDOproducing microbial organisms provided herein can initiate synthesisfrom an intermediate. The same holds true for intermediates in theformaldehyde assimilation.

In one embodiment, the carbon source is methanol or formate. In certainembodiments, methanol is used as a carbon source. In one embodiment, thecarbon source is methanol or formate. In other embodiments, formate isused as a carbon source. In specific embodiments, methanol is used as acarbon source in the organisms provided herein, either alone or incombination with the product pathways provided herein.

In one embodiment, the carbon source comprises methanol, and sugar(e.g., glucose) or a sugar-containing biomass. In another embodiment,the carbon source comprises formate, and sugar (e.g., glucose) or asugar-containing biomass. In one embodiment, the carbon source comprisesmethanol, formate, and sugar (e.g., glucose) or a sugar-containingbiomass. In specific embodiments, the methanol or formate, or both, inthe fermentation feed is provided as a mixture with sugar (e.g.,glucose) or sugar-comprising biomass. In certain embodiments, sugar isprovided for sufficient strain growth.

In some embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of from 200:1 to 1:200. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of from 100:1 to 1:100. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of from 100:1 to 5:1. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of from 50:1 to 5:1. In certainembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 100:1. In one embodiment,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 5:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 2:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio ofmethanol to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of methanol to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises formate and a sugar(e.g., glucose). In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from200:1 to 1:200. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 1:100. In some embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of formate to sugar of from 50:1 to 5:1.In certain embodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 100:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 90:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 80:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 70:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 60:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 50:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 40:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 30:1. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 20:1.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 10:1. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 5:1. In one embodiment, the sugar (e.g., glucose) isprovided at a molar concentration ratio of formate to sugar of 2:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:1. In certain embodiments,the sugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:100. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:90.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:80. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:70. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:60.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:50. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:40. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:30.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:20. In one embodiment, thesugar (e.g., glucose) is provided at a molar concentration ratio offormate to sugar of 1:10. In one embodiment, the sugar (e.g., glucose)is provided at a molar concentration ratio of formate to sugar of 1:5.In one embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of formate to sugar of 1:2. In certain embodimentsof the ratios provided above, the sugar is a sugar-containing biomass.

In certain embodiments, the carbon source comprises a mixture ofmethanol and formate, and a sugar (e.g., glucose). In certainembodiments, sugar is provided for sufficient strain growth. In someembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of from 200:1 to1:200. In some embodiments, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of from 100:1to 1:100. In some embodiments, the sugar (e.g., glucose) is provided ata molar concentration ratio of methanol and formate to sugar of from100:1 to 5:1. In some embodiments, the sugar (e.g., glucose) is providedat a molar concentration ratio of methanol and formate to sugar of from50:1 to 5:1. In certain embodiments, the sugar (e.g., glucose) isprovided at a molar concentration ratio of methanol and formate to sugarof 100:1. In one embodiment, the sugar (e.g., glucose) is provided at amolar concentration ratio of methanol and formate to sugar of 90:1. Inone embodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 80:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 70:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 60:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 50:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 40:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 30:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 20:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 10:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 5:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 2:1. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:1. In certainembodiments, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:100. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:90. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:80. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:70. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:60. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:50. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:40. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:30. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:20. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:10. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:5. In oneembodiment, the sugar (e.g., glucose) is provided at a molarconcentration ratio of methanol and formate to sugar of 1:2. In certainembodiments of the ratios provided above, the sugar is asugar-containing biomass.

1. An engineered cell either (a) expressing a non-natural NAD⁺-dependentalcohol dehydrogenase comprising at least one amino acid substitution ascompared to a corresponding alcohol dehydrogenase and capable of atleast two fold greater conversion of methanol or ethanol to formaldehydeor acetaldehyde, respectively, as compared to an engineered cellexpressing the corresponding alcohol dehydrogenase without amino acidsubstitution or (b) expressing a first sequence that is a non-naturalNAD⁺-dependent alcohol dehydrogenase comprising at least one amino acidsubstitution capable of at least two fold greater conversion of methanolor ethanol to formaldehyde or acetaldehyde, respectively, as compared toan engineered cell expressing a second sequence that is a non-naturalNAD⁺-dependent alcohol dehydrogenase, wherein the first and secondsequences differ with regards to the at least one amino acidsubstitution.

2. The engineered cell of embodiment 1 further comprising one or moremetabolic pathway transgene(s) encoding a protein of a metabolic pathwaythat promotes production of a target product or intermediate thereof.

3. The engineered cell of embodiments 1 or 2, wherein expression of thenon-natural alcohol dehydrogenase provides an increased amount ofreducing equivalents for an increase in a target product and/or forincreased fixation of carbon from the formaldehyde into a targetproduct.

4. The engineered cell of embodiment any of the previous embodimentsfurther comprising a transgene encoding an enzyme to convert theformaldehyde to formate thereby generating reducing equivalents usefulto product the target product and/or able to fix carbon of formate intothe target product.

5. The engineered cell of any of the previous embodiments wherein thetarget product is selected from the group consisting of a diol,1,4-butadiol, 1,3-butadiol, butadiene, succinate, adipate, HMDA,6-aminocaproic acid (6ACA), or an intermediate compound thereof.

6. The engineered cell of any of the previous embodiments furthercomprising one or more alcohol metabolic pathway gene(s) encoding aprotein selected from the group consisting of a), a formatedehydrogenase (EM8), a formaldehyde activating enzyme (EM10), aformaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathionesynthase (EM12), a glutathione-dependent formaldehyde dehydrogenase(EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase(EM15), and a hydrogenase (EM16).

7. The engineered cell of any of the previous embodiments furthercomprising one or more alcohol metabolic pathway gene(s) encoding aprotein selected from the group consisting of a succinyl-CoA reductase(aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase(EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), asuccinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoAtransferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HBreductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming)(EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase(EB2A).

8. A composition comprising the cell of any of the previous embodiments,or a cell extract thereof.

9. The composition of embodiment 8 wherein the composition is a cellculture composition, optionally comprising a target product orintermediate thereof.

10. A cell culture composition comprising a target product orintermediate thereof produced by the cell of any of the previousembodiments.

11. A composition comprising a target product or intermediate thereofproduced by the cell of any of the previous embodiments, optionallycomprising cell debris and/or residual culture medium.

12. The composition of embodiment 11 comprising target product orintermediate thereof. which is at least 50%, 60%, 70%, 80%, 90%, 95%,96, 97, 98, 99 or 99.9% pure in the composition.

13. The composition of embodiment 11 or 12 comprising a detectable traceamount of a nucleic acid encoding the non-natural NAD⁺-dependent alcoholdehydrogenase, or a detectable trace amount of a metabolic pathwayintermediate or product not produced in the corresponding original cellabsent expression of the non-natural NAD+-dependent alcoholdehydrogenase.

14. The composition of embodiment 11 wherein the metabolic pathwayintermediate or product is 4-hydroxybutyrate (4-HB) and 1,3 propanediol(1,3-PDO).

15. The composition of embodiment 14 comprising an amount of1,4-butanediol or 1,3-butanediol in the range of 70-90% (vol/vol) and anamount of water in the range of 10-30% (vol/vol).

16. A method for increasing the conversion of methanol or ethanol toformaldehyde or acetaldehyde, respectively, comprising a step of (a)culturing an engineered cell expressing a NAD⁺-dependent non-naturalalcohol dehydrogenase comprising at least one amino acid substitution ascompared to a corresponding alcohol dehydrogenase in a culture mediumcomprising methanol or ethanol, where in said culturing the cellprovides at least two fold greater conversion of the methanol or ethanolto formaldehyde or acetaldehyde respectively, as compared to anengineered cell expressing the corresponding alcohol dehydrogenasewithout amino acid substitution.

17. A method for increasing the conversion of methanol or ethanol toformaldehyde or acetaldehyde, respectively, comprising a step of (a)providing a reaction composition having a pH in the range of 6-8, thecomposition comprising a NAD⁺-dependent non-natural alcoholdehydrogenase comprising at least one amino acid substitution ascompared to a corresponding alcohol dehydrogenase and methanol orethanol, where in the composition said NAD⁺-dependent non-naturalalcohol dehydrogenase provides at least two fold greater conversion ofmethanol or ethanol to a formaldehyde or acetaldehyde respectively, ascompared to the corresponding alcohol dehydrogenase without amino acidsubstitution.

18. A nucleic acid encoding a NAD⁺-dependent non-natural alcoholdehydrogenase comprising at least one amino acid substitution ascompared to a corresponding alcohol dehydrogenase capable, whenexpressed in a cell, of at least two fold greater conversion of methanolor ethanol to formaldehyde or acetaldehyde respectively, as compared tothe corresponding alcohol dehydrogenase without amino acid substitution.

19. An expression construct comprising the nucleic acid of embodiment18.

20. A NAD⁺-dependent non-natural alcohol dehydrogenase comprising atleast one amino acid substitution as compared to a corresponding alcoholdehydrogenase capable, when expressed in a cell, of at least two foldgreater conversion of a methanol or ethanol to formaldehyde oracetaldehyde respectively, as compared to the corresponding alcoholdehydrogenase without amino acid substitution.

21. The subject matter of any of the previous embodiments wherein themethanol is converted to formaldehyde.

22. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase is capable of at least three foldgreater, of at least four fold, of at least five fold, of at least sixfold, of at least seven fold, at least 8 fold, at least 9 fold, at least10 fold, or at least 11 fold, conversion of methanol or ethanol to aformaldehyde or acetaldehyde, respectively, in vivo, as compared to thecorresponding alcohol dehydrogenase without amino acid substitution.

23. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase is capable of at least three foldgreater, of at least four fold, of at least five fold, of at least sixfold, of at least seven fold, at least 8 fold, at least 9 fold, at least10 fold, or at least 11 fold, conversion of methanol or ethanol to aformaldehyde or acetaldehyde, respectively, in vitro, as compared to thecorresponding alcohol dehydrogenase without amino acid substitution.

24. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase is capable of an increase inconversion of methanol or ethanol to a formaldehyde or acetaldehyderespectively, as compared to the corresponding alcohol dehydrogenasewithout amino acid substitution, in the range of two fold to twelve foldgreater, in the range of two fold to eleven fold greater, in the rangeof two fold to ten fold greater, in the range of two fold to nine foldgreater, in the range of two fold to eight fold greater, in the range oftwo fold to seven fold greater, in the range of two fold to six foldgreater, in the range of two fold to five fold greater, or in the rangeof two fold to four fold greater.

25. A NAD⁺-dependent non-natural alcohol dehydrogenase comprising atleast one amino acid substitution as compared to a corresponding alcoholdehydrogenase having a catalytic efficiency (k_(cat)/K_(m)) for theconversion of methanol to formaldehyde of 8.6×10⁻⁴ or greater.

26. The subject matter of any of the previous embodiments comprising anactivator protein that is an Act Nudix hydrolase.

27. A method of producing a target product or its intermediatecomprising culturing the engineered cell of embodiment any ofembodiments 1-7 in a culture medium comprising methanol or ethanol toproduce the the target product (TP) or its intermediate (INT).

28. The method of embodiment 27 further comprising a step of isolatingor purifying target product (TP) or its intermediate (INT).

29. The method of embodiment 28 wherein the step of isolating orpurifying comprises one or more of continuous liquid-liquid extraction,pervaporation, evaporation, filtration, membrane filtration (includingreverse osmosis, nanofiltration, ultrafiltration, and microfiltration),membrane filtration with diafiltration, membrane separation, reverseosmosis, electrodialysis, distillation, extractive distillation,reactive distillation, azeotropic distillation, crystallization andrecrystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, carbon adsorption, hydrogenation, and ultrafiltration.

30. The method of embodiment 29 selected from the group consisting of:(a) target product: 1,4-butanediol, purification: distillation; (b)target product: 1,3-butanediol, purification: distillation; (c) targetproduct: Butadiene, purification: distillation; (d) target product:6-AminoCaproic Acid, purification: crystallization, (a) target product:caprolactam, purification: distillation as a final step; (e) targetproduct: hexamethylenediame (HMDA), purification: crystallization; (f)target product: Adipic acid, purification: crystallization (adipic acidcrystals); (g) target product: Crotyl alcohol, purification:distillation (h) target product: methyl vinyl carbinol, purification:distillation; (i) target product: succinic acid—crystallization(succinic acid crystals); (j) target product: n-propanol, purification:distillation; (k) target product: isopropanol, purification:distillation; (l) target product: propylene, purification: distillation;(m) target product: methacrylic acid, purification: crystallization,distillation, or extraction (n) target product: methylmethacrylate (MMA)or another ester, purification: distillation or crystallization.

31. The method of any of embodiments 31 wherein the step of isolating orpurifying further comprises distillation.

32. The method of embodiments 28-31 wherein the target product is adiol.

33. The method of embodiments 28-32 wherein the target product is a diolis 1,4-butanediol or 1,3-butanediol.

34. The method of embodiments 28-32 comprising purifying the targetproduct to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or99.9% purity in a composition.

35. A method of preparing a polymer comprising obtaining a targetproduct produced by the engineered cell of any of embodiments 1-7 ormethod of any of the embodiments 27-34 and polymerizing the targetproduct, optionally with one or more other monomeric compounds, toprovide a polymeric product.

36. The method of embodiment 35 further comprising a step of isolatingor purifying the polymeric product.

37. The method of embodiments 35 or 36 comprising purifying the polymerproduct to at least 50%, 60%, 70%, 80%, 90%, 95%, 96, 97, 98, 99 or99.9% purity in a composition.

38. A polymer prepared according to the method of any of embodiments35-37.

39. The polymer of embodiment 38 which is a homopolymer or copolymer.

40. The polymer of embodiment 39 that is selected from the groupconsisting of polybutylene terephthalate (PBT) and polybutylenesuccinate (PBS).

41. A composition comprising a polymer blend comprising the polymer ofany ones of embodiments 38-40.

42. An article comprising the polymer or composition any one ofembodiments 38-41.

43. The article of embodiment 42 which is a plastic article.

44. The article of embodiment 17d or 17e which is molded, extruded, orshaped from the polymer or composition any one of embodiments 41-43.

45. A biobased product comprising target product produced by theengineered cell of any of embodiments 1-7 or the polymer of any ones ofembodiments 38-40 wherein said biobased product is

-   -   (i) a polymer, THF or a THF derivative, or GBL or a GBL        derivative;    -   (ii) a plastic, elastic fiber, polyurethane, polyester,        polyhydroxyalkanoate, poly-4-HB, co-polymer of poly-4-HB,        poly(tetramethylene ether) glycol, polyurethane-polyurea        copolymer, spandex, elastane, Lycra™, or nylon;    -   (iii) a polymer, a resin, a fiber, a bead, a granule, a pellet,        a chip, a plastic, a polyester, a thermoplastic polyester, a        molded article, an injection-molded article, an injection-molded        part, an automotive part, an extrusion resin, an electrical part        and a casing; and optionally where the biobased product is        reinforced or filled and further where the biobased product is        glass-reinforced or -filled or mineral-reinforced or -filled;    -   (iv) a polymer, wherein the polymer comprises polybutylene        terephthalate (PBT);    -   (v) a polymer, wherein the polymer comprises PBT and the        biobased product is a resin, a fiber, a bead, a granule, a        pellet, a chip, a plastic, a polyester, a thermoplastic        polyester, a molded article, an injection-molded article, an        injection-molded part, an automotive part, an extrusion resin,        an electrical part and a casing; and optionally where the        biobased product is reinforced or filled and further where the        biobased product is glass-reinforced or -filled or        mineral-reinforced or -filled;    -   (vi) a THF or a THF derivative, wherein the THF derivative is        polytetramethylene ether glycol (PTMEG), a polyester ether        (COPE) or a thermoplastic polyurethane;    -   (viii) a THF derivative, wherein the THF derivative comprises a        fiber; or    -   (ix) a GBL or a GBL derivative, wherein the GBL derivative is a        pyrrolidone;    -   wherein said biobased product optionally comprises at least 5%,        at least 10%, at least 20%, at least 30%, at least 40% or at        least 50% bioderived BDO; and/or wherein said biobased product        optionally comprises a portion of said bioderived BDO as a        repeating unit.

46. A molded product obtained by molding the biobased product ofembodiment 10.

47. A process for producing the biobased product of embodiment 45,comprising chemically reacting said bioderived BDO with itself oranother compound in a reaction that produces said biobased product.

48. A polymer comprising or obtained by converting the bioderived BDO ofembodiment 45.

49. A method for producing a polymer, comprising chemically ofenzymatically converting the bioderived BDO of embodiment 45 to thepolymer.

50. A composition comprising the bioderived BDO of embodiment 45, or acell lysate or culture supernatant thereof.

51. A method of producing an intermediate of glycolysis and/or anintermediate of a metabolic pathway that can be used in the formation ofbiomass, comprising culturing the engineered cell of any one ofembodiments 1-7 under conditions and for a sufficient period of time toproduce the intermediate, and optionally wherein the intermediate isconsumed to provide a reducing equivalent or to incorporate into BDO ortarget product.

52. The method of embodiment 51, wherein the organism is cultured in amedium comprising biomass, glucose, xylose, arabinose, galactose,mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide,formate, methane, or any combination thereof as a carbon source.

53. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to an NAD⁺-dependent-alcoholdehydrogenase template selected from the group consisting of SEQ ID NO:1(MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1,EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1,YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161,YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1,YP_007491369.1, YP_002434746, YP_005052855, NP_561852.1, YP_001447544,YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1,NP_717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1,YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or afragment of said template having said dehydrogenase activity with anamino-terminal deletion, carboxy-terminal deletion, or both, thefragment having a sequence identity of 45% or greater, 55% or greater,65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% orgreater, 95% or greater, 96% or greater, 97% or greater, 98% or greater,or 99% or greater to the template.

54. The subject matter of any of the previous embodiments said templateis selected from the group consisting of EIJ77596.1, EIJ78397.1,EFI69743.1, YP_001699778.1, YP_002138168.1, YP_359772.1, YP_005052855,NP_561852.1, YP_001447544, ZP_01220157.1, YP_003990729.1, ZP_10241531.1,and YP_026233.1.

55. The subject matter of any of the previous embodiments wherein thealcohol dehydrogenase is a methanol dehydrogenase.

56. The subject matter of embodiment 55 wherein the methanoldehydrogenase is from bacteria.

57. The subject matter of embodiment 56 wherein the methanoldehydrogenase is from Bacillus.

58. The subject matter of embodiment 57 wherein the methanoldehydrogenase is from Bacillus methanolicus MGA3 or Bacillusmethanolicus

59. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase has a sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and wherein the dehydrogenase comprises one or moreamino acid substitutions based on formula: R¹XR², where R¹ is anoriginal amino acid at position X of the template, and R² is the variantamino acid that replaces R¹ at a position on the template correspondingto X, wherein XR² is selected from the group consisting of (a) 11T, 38N,42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G,84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K,112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L,121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L,123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R,128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T,145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M,149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A,161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R,184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S,270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D,344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K,360R, 360S, 361N, 361R, 363K, and 379M or group consisting of (b) 38N,60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D,117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P,123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G,127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I,130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H,158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K,360R, 360S, and 361R.

60. The subject matter of embodiment 59 wherein XR² is selected from thegroup consisting of 107S, 121D, 123D, 123I, 123L, 123R, 123Y, 129A,129M, 129P, 129S, 129V, 130F, 130I, 130Y, 143T, 146N, 149L, 149M, 149T,149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y, 355D, 360G, 360K,360R, and 360S

61. The subject matter of embodiment 60 wherein R¹XR² is selected fromthe group consisting of (a) S11T, D38N, H42Q, E48D, N53I, E56K, D60E,V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R, E86K, N87K,I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y, N112K,N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A, G121D,G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122A,V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R, V125C,V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S, V129A,V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M, T146N,S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L, A149M,A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H, V158K,V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q, I163T,D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V, Q217K,L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H, A300T,I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A, V344G,K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G, V360K,V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E, P71I,P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F,N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V,G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L,V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A,V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L,A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q,D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R,V360S, and C361R.

62. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and comprises a original amino acid at all positionsthat are not substituted at amino acid position numbers of group (a) 11,38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99,103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149,150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199,217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333,336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; orof group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150,157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.

63. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and comprises a original amino acid at all positionsthat are not amino acid position numbers 107, 121, 123, 129, 130, 143,146, 149, 158, 267, 270, 355, 360.

64. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises two, three, four, five, six,seven, eight, nine, ten, eleven or twelve, amino acid substitutionsselected from the group consisting of: (a) S11T, D38N, H42Q, E48D, N53I,E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R,E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y,N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A,G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y,V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R,V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S,V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M,T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L,A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H,V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q,I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V,Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H,A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A,V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G,V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or (b) D38N, D60E,P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W, V109Y, R115H, I116F,N117D, N117Q, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V,G121W, G121Y, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L,V125C, V125G, V125W, E126G, K127C, K127R, P128A, P128R, P128S, V129A,V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T146N, A149L,A149M, A149T, A149V, V150A, K157N, V158E, V158H, V158K, V158W, I163Q,D164N, Q267H, G270M, G270S, G270Y, K345E, N355D, V360G, V360K, V360R,V360S and C361R.

65. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a set of amino acidsubstitutions selected from the group consisting of (a) D70N, L148G,P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I,P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N,P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I,P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (j) L148G, P161A,V360A, C361N; (k) L148G, P161A, V360G; (l) L148G, P161A, V360G, C361N;(m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G,V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r)L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u)L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I,P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I,P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V,V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W,P161A, V360A, C361N; (ad) N112K, S147R, P161A, V360A; (ae) P161A, Q217K,V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V,E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N;(ak) N112K, S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am)P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap)P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A,V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F,V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G;(aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I,P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V,P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V,V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad)S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R,P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G,C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A,C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N;(aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao)V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I,P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I,P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I,V360A, C361N; (aav) V150I, V360G; (aaw) S11T, T74S, G269S, V344A; (aax)K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M,T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K,S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q,G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L;(aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G;(aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I;(aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V;(aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K,N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay)G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y,T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R;(aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai)P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal)D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161S,and (aaaap) N112R/P161S.

66. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TNA and VTNAF (SEQ ID NO: 79).

67. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of VEV and GVEVA (SEQ ID NO: 80).

68. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD(SEQ ID NO: 82).

69. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of EKC and QEKCD (SEQ ID NO: 83).

70. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of STH and GSTHD (SEQ ID NO: 84).

71. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TVK and DTVKA (SEQ ID NO: 85).

72. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of SLV, GVV, GWV, GLY, ISLVA (SEQ ID NO: 86),IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89)

73. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of HIN, RFN, RID, RIQ, GHIND (SEQ ID NO: 90),GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93).

74. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97),RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPVV (SEQ ID NO: 99), TVNSVEKPVV (SEQID NO: 100), VVNSVEKPVV (SEQ ID NO: 101), WVNSVEKPVV (SEQ ID NO: 102),YVNSVEKPVV (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPVV(SEQ ID NO: 105), GVISVEKPVV (SEQ ID NO: 106), GVLSVEKPVV (SEQ ID NO:107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPVV (SEQ ID NO: 109).GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPVV (SEQ ID NO: 111), GVNSCEKPVV(SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO:114), GVNSVGKPVV (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116),GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAVV (SEQ ID NO: 118). GVNSVEKRVV(SEQ ID NO: 119). GVNSVEKSVV (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO:121). GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123).GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI(SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127).

75. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO:129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131).

76. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO:133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO:136).

77. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ IDNO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID(SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO:143).

78. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO:145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147).

79. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of VEE and GVEEE (SEQ ID NO: 148).

80. The subject matter of any of the previous embodiments wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ IDNO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), andNAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154).

81. A nucleic acid encoding the non-natural alcohol dehydrogenase of anyof embodiments 53-80.

82. A non-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to an NAD⁺-dependent alcoholdehydrogenase template selected from the group consisting of SEQ ID NO:1(MDH MGA3_17392), EIJ77596.1, AAA22593.1, EIJ77618.1, EIJ78790.1,EIJ80770.1, EIJ78397.1, EIJ83020.1, EFI69743.1, YP_004860127.1,YP_001699778.1, ZP_11313277.1, ZP_05587334.1, YP_004681552.1, AGF87161,YP_002138168.1, YP_359772.1, YP_001343716.1, ZP_16224338.1, AAC45651.1,YP_007491369.1, YP_002434746, YP_005052855, NP_561852.1, YP_001447544,YP_001113612.1, YP_011618, ZP_01220157.1, YP_003990729.1, ZP_07335453.1,NP_717107, YP_003310546.1, ZP_10241531.1, YP_001337153.1, YP_026233.1,YP_694908, YP_725376.1, YP_001663549, EKC54576, YP_001126968.1 or afragment of said template having said dehydrogenase activity with anamino-terminal deletion, carboxy-terminal deletion, or both, thefragment having a sequence identity of 45% or greater, 55% or greater,65% or greater, 75% or greater, 85% or greater, 90% or greater, 92.5% orgreater, 95% or greater, 96% or greater, 97% or greater, 98% or greater,or 99% or greater to the template.

83. A non-natural alcohol dehydrogenase of embodiment 82 wherein saidtemplate is selected from the group consisting of EIJ77596.1,EIJ78397.1, EFI69743.1, YP_001699778.1, YP_002138168.1, YP_359772.1,YP_005052855, NP_561852.1, YP_001447544, ZP_01220157.1, YP_003990729.1,ZP_10241531.1, and YP_026233.1.

84. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase has a sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and wherein the dehydrogenase comprises one or moreamino acid substitutions based on formula: R¹XR², where R¹ is a originalamino acid at position X of the template, and R² is the variant aminoacid that replaces R¹ at a position on the template corresponding to X,wherein XR² is selected from the group consisting of (a) 11T, 38N, 42Q,48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R,86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R,115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M,121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R,123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A,128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M,146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T,149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G,161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T,186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y,296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A,344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R,360S, 361N, 361R, 363K, and 379M or the group consisting of (b) 38N,60E, 71I, 71V, 87K, 99T, 103V, 107S, 108V, 108W, 109Y, 115H, 116F, 117D,117Q, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122P,123D, 123I, 123L, 123R, 123Y, 124I, 124L, 125C, 125G, 125V, 125W, 126G,127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 129V, 130F, 130I,130Y, 134T, 143T, 146N, 149L, 149M, 149T, 149V, 150A, 157N, 158E, 158H,158K, 158W, 163Q, 164N, 267H, 270M, 270S, 270Y, 345E, 355D, 360G, 360K,360R, 360S, and 361R.

85. A non-natural alcohol dehydrogenase of embodiment 82 wherein XR² isselected from the group consisting of 107S, 121D, 123D, 123I, 123L,123R, 123Y, 129A, 129M, 129P, 129S, 129V, 130F, 130I, 130Y, 143T, 146N,149L, 149M, 149T, 149V, 158E, 158H, 158K, 158W, 267H, 270M, 270S, 270Y,355D, 360G, 360K, 360R, and 360S

86. A non-natural alcohol dehydrogenase of embodiment 82 wherein R¹XR²is selected from the group consisting of (a) S11T, D38N, H42Q, E48D,N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G,K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W,V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R,G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W,G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L,S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R,P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T,T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W,A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E,V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N,I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S,I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S,R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D,V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A,V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379M; or the groupconsisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S,L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L,G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I,N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C,K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I,V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N,V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y,K345E, N355D, V360G, V360K, V360R, V360S, and C361R.

87. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and comprises an original amino acid at all positionsthat are not substituted at amino acid position numbers of group (a) 11,38, 42, 48, 53, 56, 60, 61, 63, 65, 70, 71, 74, 81, 84, 86, 87, 94, 99,103, 106, 107, 108, 109, 112, 115, 116, 117, 117, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 134, 143, 145, 146, 147, 148, 149,150, 152, 155, 157, 158, 161, 163, 164, 165, 181, 184, 186, 190, 199,217, 226, 256, 267, 269, 270, 296, 298, 300, 302, 312, 316, 323, 333,336, 337, 343, 344, 345, 350, 354, 355, 358, 360, 361, 363 and 379; orof group (b) 38, 60, 71, 87, 99, 103, 107, 108, 109, 115, 116, 117, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 143, 146, 149, 150,157, 158, 163, 164, 267, 270, 345, 355, 360, and 361.

88. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase has sequence identity of 45% orgreater, 55% or greater, 65% or greater, 75% or greater, 85% or greater,90% or greater, 92.5% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, or 99% or greater to any one of the templatesof embodiment 53, and comprises an original amino acid at all positionsthat are not amino acid position numbers 107, 121, 123, 129, 130, 143,146, 149, 158, 267, 270, 355, 360.

89. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises two, three, four, five, six,seven, eight, nine, ten, eleven or twelve amino acid substitutionsselected from the group consisting of: (a) S11T, D38N, H42Q, E48D, N53I,E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V, T74S, D81G, K84R,E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S, L108V, L108W, V109Y,N112K, N112R, R115H, I116F, N117D, N117Q, N117Y, Q120H, Q120R, G121A,G121D, G121E, G121L, G121M, G121R, G121S, G121T, G121V, G121W, G121Y,V122A, V122P, N123D, N123I, N123L, N123R, N123Y, S124I, S124L, S124R,V125C, V125G, V125W, E126G, E126V, K127C, K127R, P128A, P128R, P128S,V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T, S143T, T145M,T146N, S147R, L148A, L148F, L148G, L148I, L148T, L148V, L148W, A149L,A149M, A149T, A149V, V150A, V150I, T152M, A155V, K157N, V158E, V158H,V158K, V158W, P161A, P161G, P161Q, P161S, P161V, I163F, I163N, I163Q,I163T, D164G, D164N, E165G, K181R, A184T, L186M, T190A, T190S, I199V,Q217K, L226M, G256C, Q267H, G269S, G270M, G270S, G270Y, T296S, R298H,A300T, I302V, G312V, A316V, I323M, F333L, P336L, S337C, G343D, V344A,V344G, K345E, E350K, K354M, N355D, N355I, N355K, E358G, V360A, V360G,V360K, V360R, V360S, C361N, C361R, Q363K, and K379M or the groupconsisting of (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S,L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L,G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I,N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C,K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I,V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N,V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y,K345E, N355D, V360G, V360K, V360R, V360S and C361R.

90. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a set of amino acidsubstitutions selected from the group consisting of (a) D70N, L148G,P161G, V360A; (b) D70N, L148G, V360A, C361N; (c) D70N, L148V, V150I,P161A, V360G; (d) D70N, L148V, V360G; (e) D70N, P161A, V360A; (f) D70N,P161V, V360G, C361N; (g) D70N, V150I, P161A, V360A; (h) D70N, V150I,P161V, V360G, C361N; (i) E48D, L148V, P161A, V360A; (j) L148G, P161A,V360A, C361N; (k) L148G, P161A, V360G; (l) L148G, P161A, V360G, C361N;(m) L148G, P161G, V360A; (n) L148G, P161G, V360G, C361N; (o) L148G,V360A, C361N; (p) L148G, V360G, C361N; (q) L148I, P161G, V360G; (r)L148I, P161V, V360G; (s) L148T, V150I, V360A; (t) L148T, V360G; (u)L148V, P161A, V360A; (v) L148V, V150I, P161A, V360A; (w) L148V, V150I,P161A, V360A, C361N; (x) L148V, V150I, P161A, V360G; (y) L148V, V150I,P161A, V360G, C361N; (z) L148V, V150I, P161A, V360G, C361N; (aa) L148V,V150I, P161G, V360A; (ab) L148V, V150I, P161V, V360G, C361N; (ac) L148W,P161A, V360A, C361N; (ad) N112K, S147R, P161A, V360A; (ae) P161A, Q217K,V360A, C361N; (af) P161A, V360A, C361N; (ag) P161A, V360G; (ah) P161V,E358G, V360G; (ai) P161V, V360A, C361N; (aj) L148W, P161A, V360A, C361N;(ak) N112K, S147R, P161A, V360A; (al) P161A, Q217K, V360A, C361N; (am)P161A, V360A, C361N; (an) P161A, V360G; (ao) P161V, E358G, V360G; (ap)P161V, V360A, C361N; (aq) P161V, V360G; (ar) P65Q, L148G, V150I, P161A,V360G, C361N; (as) S147R, L148A, V150I, P161A, V360G; (at) S147R, L148F,V150I, P161G, V360G; (au) S147R, L148V, P161G, V360A; (av) P161V, V360G;(aw) P65Q, L148G, V150I, P161A, V360G, C361N; (ax) S147R, L148A, V150I,P161A, V360G; (ay) S147R, L148F, V150I, P161G, V360G; (az) S147R, L148V,P161G, V360A; (aaa) S147R, L148V, P161V, V360G; (aab) S147R, L148V,V150I, P161A, C361N; (aac) S147R, L148V, V150I, P161G, V360G; (aad)S147R, P161A, V360A; (aae) S147R, P161A, V360A, C361N; (aaf) S147R,P161A, V360G; (aag) S147R, P161V, V360G; (aah) S147R, P161V, V360G,C361N; (aai) S147R, V150I, P161V, V360A; (aaj) S147R, V150I, V360A,C361N; (aak) T145M, L148I, V360G; (aal) V150I, I302V, V360G, C361N;(aam) V150I, P161A, C361N; (aan) V150I, P161G, V360A, C361N; (aao)V150I, P161G, V360G; (aap) V150I, P161G, V360G, C361N; (aaq) V150I,P161V, C361N; (aar) V150I, P161V, K354R, V360A, C361N; (aas) V150I,P161V, V360A, C361N; (aat) V150I, P161V, V360G, C361N; (aau) V150I,V360A, C361N; (aav) V150I, V360G; (aaw) S11T, T74S, G269S, V344A; (aax)K84R, I163T; (aay) V122A, I163N; (aaz) G107S, F333L; (aaaa) V129M,T152M, G343D; (aaab) I63F, N355K; (aaac) G107S, F333L; (aaad) E86K,S99T, A149V; (aaae) N53I, V158E; (aaaf) N355I, K379M; (aaag) H42Q,G107S; (aaah) Q120H, I163N; (aaai) A149V, I323M; (aaaj) G107S, F333L;(aaak) D164G, K181R; (aaal) A155V, R298H, N355D; (aaam) N123D, E165G;(aaan) I163F, L186M; (aaao) G121A, T296S; (aaap) I94V, S99P, N123I;(aaaq) E126V, V129M, V344G; (aaar) Q120R, S143T; (aaas) G256C, A316V;(aaat) P161Q, G312V; (aaau) L226M, A300T, V360A; (aaav) S337C, E350K,N355D, Q363K; (aaaw) D81G, V158E; (aaax) I106L, N117Y, E126V; (aaay)G107S, G121D; (aaaz) V61A, V158E; (aaaaa) N53I, V158E; (aaaab) N117Y,T190S; (aaaac) S124R, I199V; (aaaad) K354M, C361R; (aaaae) A184T, C361R;(aaaag) E56K, Q267H; (aaaag) S124R, E126G; (aaaah) T190A, N355K; (aaaai)P71T, F333L; (aaaaj) G107S, F333L; and (aaaak) N123I, P336L, (aaaal)D38D/A149V, (aaaam) D38N/V163V, (aaaan) D73D/L108V, (aaaao) G121R/P161S,and (aaaap) N112R/P161S.

91. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TNA and VTNAF (SEQ ID NO: 79).

92. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of VEV and GVEVA (SEQ ID NO: 80).

93. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD(SEQ ID NO: 82).

94. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of EKC and QEKCD (SEQ ID NO: 83).

95. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of STH and GSTHD (SEQ ID NO: 84).

96. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TVK and DTVKA (SEQ ID NO: 85).

97. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of SLV, GVV, GWV, GLY, ISLVA (SEQ ID NO: 86),IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA (SEQ ID NO: 89).

98. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of HIN, RFN, RID, RIQ, GHIND (SEQ ID NO: 90),GRFND (SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93).

99. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQID NO: 95), LVNSVEKPVV (SEQ ID NO: 96), MVNSVEKPVV (SEQ ID NO: 97),RVNSVEKPVV (SEQ ID NO: 98), SVNSVEKPVV (SEQ ID NO: 99), TVNSVEKPVV (SEQID NO: 100), VVNSVEKPVV (SEQ ID NO: 101), WVNSVEKPVV (SEQ ID NO: 102),YVNSVEKPVV (SEQ ID NO: 103), GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPVV(SEQ ID NO: 105), GVISVEKPVV (SEQ ID NO: 106), GVLSVEKPVV (SEQ ID NO:107), GVRSVEKPVV (SEQ ID NO: 108), GVYSVEKPVV (SEQ ID NO: 109).GVNIVEKPVV (SEQ ID NO: 110), GVNLVEKPVV (SEQ ID NO: 111), GVNSCEKPVV(SEQ ID NO: 112), GVNSGEKPVV (SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO:114), GVNSVGKPVV (SEQ ID NO: 115), GVNSVECPVV (SEQ ID NO: 116),GVNSVERPVV (SEQ ID NO: 117), GVNSVEKAVV (SEQ ID NO: 118). GVNSVEKRVV(SEQ ID NO: 119). GVNSVEKSVV (SEQ ID NO: 120). GVNSVEKPAV (SEQ ID NO:121). GVNSVEKPMV (SEQ ID NO: 122). GVNSVEKPPV (SEQ ID NO: 123).GVNSVEKPSV (SEQ ID NO: 124). GVNSVEKPVF (SEQ ID NO: 125). GVNSVEKPVI(SEQ ID NO: 126), and GVNSVEKPVY (SEQ ID NO: 127).

100. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of TETT (SEQ ID NO: 128), SETN (SEQ ID NO:129), GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131).

101. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of LLVI (SEQ ID NO: 132), LMVI (SEQ ID NO:133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO: 135), and LAAI (SEQ ID NO:136).

102. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of NVKMPVID (SEQ ID NO: 137), KEKMPVID (SEQ IDNO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140), KWKMPVID(SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQ ID NO:143).

103. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO:145), QVGS (SEQ ID NO: 146), and QVGY (SEQ ID NO: 147).

104. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of VEE and GVEEE (SEQ ID NO: 148).

105. A non-natural alcohol dehydrogenase of embodiment 82 wherein thenon-natural alcohol dehydrogenase comprises a sequence motif selectedfrom the group consisting of DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ IDNO: 150), NAYEDKC (SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), andNAYEDSC (SEQ ID NO: 153), and NAYEDVR (SEQ ID NO: 154).

106. A nucleic acid encoding the non-natural alcohol dehydrogenase ofany of embodiments 82-105.

107. An expression construct comprising the nucleic acid of 106.

108. An engineered cell comprising the nucleic acid or expressionconstruct of embodiments 106 or 107.

109. The engineered cell of embodiment 108 further comprising one ormore alcohol metabolic pathway gene(s) encoding a protein selected fromthe group consisting of a), a formate dehydrogenase (EM8), aformaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase(EM11), a S-(hydroxymethyl)glutathione synthase (EM12), aglutathione-dependent formaldehyde dehydrogenase (EM13), aS-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), ahydrogenase (EM16).

110. The engineered cell of embodiment 108 further comprising one ormore alcohol metabolic pathway gene(s) encoding a protein selected fromthe group consisting of a succinyl-CoA reductase (aldehyde forming)(EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase(EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoAreductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase(EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcoholforming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoAtransferase (EB1), and a succinyl-CoA synthetase (EB2A).

111. The engineered cell of embodiment 108-110 which is bacteria.

112. The transgenic bacteria of embodiment 111 which is Bacillus.

113. A method for increasing the conversion of a methanol or ethanol toa dehydrogenated product of the alcohol comprising a step of (a)culturing the engineered cell of any of embodiments 108-112 in a culturemedium comprising a methanol or ethanol, where in said culturing thecell provides at least two fold greater conversion of the methanol orethanol to a dehydrogenated product of the alcohol, as compared to anengineered cell expressing a corresponding alcohol dehydrogenase withoutamino acid substitution.

114. A method for increasing the conversion of a methanol or ethanol toa dehydrogenated product of the alcohol comprising a step of (a)providing a reaction composition having a pH in the range of 6-8, thecomposition comprising a non-natural alcohol dehydrogenase of any ofembodiments 82-105, where in the composition said culturing the cellprovides at least two fold greater conversion of the methanol or ethanolto a dehydrogenated product of the alcohol, as compared to an engineeredcell expressing a corresponding alcohol dehydrogenase without amino acidsubstitution.

115. A method of providing a diol comprising culturing the engineeredcell of any of embodiments 108-112 in a culture medium comprising amethanol or ethanol to provide the diol.

116. The method of embodiment 115 wherein the diol is 1,4 butanediol.

117. A method of preparing a polymer comprising obtaining a monomerproduct produced by the engineered cell or method of any of embodiments108-116 and polymerizing the monomer to provide a polymeric product.

118. A polymer prepared according to the method of embodiment 117.

119. A method of screening for a non-natural alcohol dehydrogenasehaving increased activity, optionally at least 2 fold, optionally atleast 4 fold or greater activity, compared to its unmodifiedcounterpart, comprising (1) creating one or more non-natural alcoholdehydrogenases selected from SEQ ID NO:1 and non-natural alcoholdehydrogenases having a sequence identity of 45% or greater to SEQ IDNO:1 having a substitution at a position other than an amino acidposition selected from group (a) 11, 38, 42, 48, 53, 56, 60, 61, 63, 65,70, 71, 74, 81, 84, 86, 87, 94, 99, 103, 106, 107, 108, 109, 112, 115,116, 117, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,134, 143, 145, 146, 147, 148, 149, 150, 152, 155, 157, 158, 161, 163,164, 165, 181, 184, 186, 190, 199, 217, 226, 256, 267, 269, 270, 296,298, 300, 302, 312, 316, 323, 333, 336, 337, 343, 344, 345, 350, 354,355, 358, 360, 361, 363 and 379; or of group (b) 38, 60, 71, 87, 99,103, 107, 108, 109, 115, 116, 117, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 134, 143, 146, 149, 150, 157, 158, 163, 164, 267, 270,345, 355, 360, and 361, or a position corresponding thereto, (2) assaythe created enzyme for the activity and (3) selecting those havingincreased activity, optionally at least 2 fold, optionally at least 4fold, or greater activity compared to the unmodified counterpart.

EXAMPLES

Assay for Testing Activity of Methanol Dehydrogenase In Vitro

A high-throughput screening assay was used to evaluate lysates formethanol dehydrogenase (MeDH) oxidation activity of methanol and otheralcohol substrates. Lysates were prepared by a commercial chemicalreagent from Escherichia coli cells that contained a plasmid harboring aMeDH library variant and an integrated chromosomal copy of the activatorprotein. An aliquot of the lysate was applied to a 384-well assay plate.To initiate the alcohol oxidation reaction, a substrate-buffer mix (pH7.6 or pH 8.5) containing 0.5 M methanol or other alcohol, 0.5 mM NAD, 5mM MgCl₂, 10 μM 1-methoxy-5-methylphenazinium methylsulfate (1-methoxyPMS), & 1 mM 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) was added. Initial rates were monitored via absorbance at560 nm. MeDH variants that showed higher activity than the wild-typecontrol were evaluated for further characterization.

Formaldehyde Assay

A strain lacking frmA, frmB, frmR (the genes responsible forformaldehyde utilization in E. coli) was created using Lamba Redrecombinase technology. Plasmids expressing methanol dehydrogenases weretransformed into the strain, then grown to saturation in LBmedium+antibiotic at 37° C. with shaking. Cultures were adjusted by ODand then diluted 1:10 into M9 medium+0.5% glucose+antibiotic andcultured at 37° C. with shaking for 6-8 h until late log phase. Methanolwas added to 2% v/v and the cultures were further incubated for 30 minwith shaking at 37° C. Cultures were spun down and the supernatant wasassayed for formaldehyde produced using DetectX Formaldehyde Detectionkit from Arbor Assays, MI according to manufacturer's instructions.

Formate Assay

The assay was developed to evaluate in vivo activity of methanoldehydrogenases by measuring the formate production in a host straincontaining the first two steps of the MeOH pathway but lacking formatedehydrogenases (hycE, fdnGHI, fdoGHI, fdhF) that convert formate to CO₂.Plasmids expressing methanol dehydrogenases were transformed into thisstrain. Strains were inoculated from colonies or glycerol stocks inLB+antibiotics in 96-deep well plates. The plates were sealed withbreathable culture films and shaken at 37° C. at 800 rpm. Overnightcultures were centrifuged at 5250 rpm for 10 minutes to pellet thecells. Cells were resuspended in 1 ml M9 medium in plates that weresealed with breathable culture films and shaken at 37 degree at 800 rpm.Samples were taken for a time course study and formate concentrationswere measured using the formate kit based on instructions provided bythe manufacturer.

Assay for Purification of Methanol Dehydrogenases and for Characterizingtheir Activity

Cells expressing methanol dehydrogenase are cultured at 37° C. in LBcontaining 2 mM MgSO₄. Once harvested, cells are lysed in BugBusterProtein Extraction Reagent (Novagen) supplemented with 15 kU/mL lysozyme(Novagen), 25 U/mL bezonase (Novagen), 1× Pierce Protease Inhibitors(Thermo Scientific), 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride,and 2 mM MgSO4. Lysates are clarified via centrifugation and purified ona 5 mL StrepTrap HP column (GE Healthcare Life Sciences). The column isprepared in and washed with 100 mM MOPS pH 7.5, 0.2 M NaCl2, 2 mM MgSO₄,0.5 mM TCEP (buffer A). The purified proteins are eluted with buffer Acontaining 0.3 mg/mL desthiobiotin.

DNA 2.0 Gene Synthesis

Methanol dehydrogenase gene candidates were synthesized after optimizingfor codon usage by DNA 2.0 (Welch et al., PloS One 2009, 4(9):e7002,Design parameters to control synthetic gene expression in Escherichiacoli).

In Vivo Labeled Assay for Conversion of Methanol to CO₂

Strains with functional reductive TCA branch and pyruvate formate lyasedeletion were grown aerobically in LB medium overnight, followed byinoculation of M9 high-seed media containing IPTG and aerobic growth for4 hrs. These strains had methanol dehydrogenase/ACT pairs in thepresence and absence of formaldehyde dehydrogenase or formatedehydrogenase. At this time, strains were pelleted, resuspended in freshM9 medium high-seed media containing 2% ¹³CH₃OH, and sealed in anaerobicvials. Head space was replaced with nitrogen and strains grown for 40hours at 37° C. Following growth headspace was analyzed for 13-CO₂.Media was examined for residual methanol as well as BDO and byproducts.

All constructs expressing MeDH mutants and MeDH/ACT pairs grew toslightly lower ODs than strains containing empty vector controls. Thisis likely due to the high expression of these constructs.

Description of the NAD-Dependent Methanol Dehydrogenase/ActivatorProtein, its Expression and Use

Sequence analysis of the NADH-dependent methanol dehydrogenase fromBacillus methanolicus places the enzyme in the alcohol dehydrogenasefamily III. It does not contain any tryptophan residues, resulting in alow extinction coefficient (18,500 M⁻¹, cm⁻¹) and should be detected onSDS gels by Coomassie staining.

The enzyme has been characterized as a multisubunit complex built from43 kDa subunits containing one Zn and 1-2 Mg atoms per subunit. Electronmicroscopy and sedimentation studies determined it to be a decamer, inwhich two rings with five-fold symmetry are stacked on top of each other(Vonck et al., J. Biol. Chem. 266, p. 3949-3954, 1991). It is describedto contain a tightly but not covalently bound cofactor and requiresexogenous NAD⁺ as e⁻-acceptor to measure activity in vitro. A strongincrease (10-40-fold) of in vitro activity was observed in the presenceof an activator protein (Act), which is a homodimer (21 kDa subunits)and contains one Zn and one Mg atom per subunit.

The mechanism of the activation was investigated by Kloosterman et al.(J. Biol. Chem. 277, p. 34785-34792, 2002), showing that Act is a Nudixhydrolase and Hektor et al. (J. Biol. Chem. 277, p. 46966-46973, 2002),demonstrating that mutation of residue S97 to G or T in MeDH changesactivation characteristics along with the affinity for the cofactor.While mutation of residues G15 and D88 had no significant impact, a roleof residue G13 for stability as well as of residues G95, D100, and K103for the activity is suggested. Both papers together propose a hypothesisin which Act cleaves MeDH-bound NAD⁺. MeDH retains AMP bound and entersan activated cycle with increased turnover.

The stoichiometric ratio between Act and MeDH is not well defined in theliterature. Kloosterman et al. (J. Biol. Chem. 277, p. 34785-34792,2002) determine the ratio of dimeric Act to decameric MeDH for full invitro activation to be 10:1. In contrast, Arfman et al. (J. Biol. Chem.266, 3955-3960, 1991) determined a ratio of 3:1 in vitro for maximum anda 1:6 ratio for significant activation, but observe a high sensitivityto dilution. Based on expression of both proteins in Bacillus, theauthors estimate the ratio in vivo to be around 1:17.5. In vitroexperiments with purified activator protein (2317A) and methanoldehydrogenase (2315A) have showed the ratio of “act” to methanoldehydrogenase to be 10:1. This in vitro test was done with 5 M methanol,2 mM NAD and 10 uM methanol dehydrogenase 2315A at pH 7.4.

The sequence of the activator protein (SEQ ID NO: 157) from Bacillusmethanolicus MGA3 (locus tag: MGA3_09170, GI number: 387591061,Accession number: EIJ83380) used in the assays is shown below:

MGKLFEEKTIKTEQIFSGRVVKLQVDDVELPNGQTSKREIVRHPGAVAVIAITNENKIVMVQYRKPLEKSIVEIPAGKLEKGEDPRITALRELEEETGYECEQMEWLISFATSPGFADEIIHIYVAKGLSKKENAAGLDEDEFVDLIELTLDEALQYIKEQRIYDSKTVIAVQYLQLQEALKNK.2315 Stability Assay and Data

The thermostability of methanol dehydrogenase 2315A and thecorresponding activator protein 2317A were assessed and meltingtemperatures were found to be 62 and 75° C., respectively. The meltingtemperatures were measured using a Protein thermal shift assay fromApplied biosystems. The assay provides relative thermal stabilities(melting temperatures) of purified proteins. It relies on a proprietaryfluorescent dye that binds to hydrophobic regions of denatured proteinsupon heating in the RT-PCR machine. The relative melting temperature iscalculated from the slope of the fluorescence signal peak.

Current Promoter and Plasmid for Overexpression

Methanol dehydrogenase 2315 was expressed with several constitutive andinducible promoters of varying strengths. The figure below shows theexpression levels of two MeDH variants when expressed under threepromoters: p119, p104 and p107. The two variants that were expressedwere 2315L and 2315B. 2315B was a mutant constituted based on a mutationS97G identified from Hektor et al (ibid).

MDH Protein Concentrations

Methanol dehydrogenase is a very soluble protein. SDS-PAGE analysis ofsoluble proteins from lysates of E. coli strains expressing differentvariants of the WT 2315A are shown. Specifically, the left panel showsthe gel run on the lysates and the right panel shows the gel run onsupernatant for the WT enzyme 2315A, compared with the variants 2315Land R, a variant from Hektor et al. called 2315B, and an empty vector.

The cells were lysed using Bugbuster as described previously. The amountof protein was quantified using the Image Lab 3.0 software from BioRad.The WT protein was estimated to be 27% of the total protein.

Background on Plasmids and Promoters

Vector backbones were obtained from Dr. Rolf Lutz of Expressys(www.expressys.de). The vectors and strains are based on the pZExpression System developed by Dr. Rolf Lutz and Prof. Hermann Bujard(Lutz, R. & Bujard, H. Independent and tight regulation oftranscriptional units in Escherichia coli via the LacR/O, the TetR/O andAraC/I1-I2 regulatory elements. Nucleic Acids Res 25, 1203-1210 (1997)).Art available promoters P119, p104, p107, p119 provided varying levelsof enzyme expression as desired. Vectors obtained were pZE13luc,pZA33luc, pZS*13luc and pZE22luc and contained the luciferase gene as astuffer fragment. To replace the luciferase stuffer fragment with alacZ-alpha fragment flanked by appropriate restriction enzyme sites, theluciferase stuffer fragment was first removed from each vector bydigestion with EcoRI and XbaI. The lacZ-alpha fragment was PCR amplifiedfrom pUC19 with the following primers:

lacZalpha-RI (SEQ ID NO: 155)5′GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGGC CGTCGTTTTAC3′lacZalpha 3′BB (SEQ ID NO: 156)5′-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAG A-3′

This generated a fragment with a 5′ end of EcoRI site, NheI site, aRibosomal Binding Site, a SalI site and the start codon. The 3′ end ofthe fragment contained the stop codon, XbaI, HindIII, and AvrII sites.The PCR product was digested with EcoRI and AvrII and ligated into thebase vectors digested with EcoRI and XbaI (XbaI and AvrII havecompatible ends and generate a non-site). Because NheI and XbaIrestriction enzyme sites generate compatible ends that can be ligatedtogether (but generate a site after ligation that is not digested byeither enzyme), the genes cloned into the vectors could be “Biobricked”together (openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly,this method enables joining an unlimited number of genes into the vectorusing the same 2 restriction sites (as long as the sites do not appearinternal to the genes), because the sites between the genes aredestroyed after each addition. Initially, expression was low from thesevectors, and they were subsequently modified using the Phusion®Site-Directed Mutagenesis Kit (NEB, Ipswich, Mass., USA) to insert thespacer sequence AATTAA between the EcoRI and NheI sites. This eliminateda putative stem loop structure in the RNA that bound the RBS and startcodon.

All vectors have the pZ designation followed by letters and numbersindicating the origin of replication, antibiotic resistance marker andpromoter/regulatory unit. The origin of replication is the second letterand is denoted by E for ColE1, A for p15A and S for pSC101 (as well as alower copy number version of pSC101 designated S*)-based origins. Thefirst number represents the antibiotic resistance marker (1 forAmpicillin, 2 for Kanamycin, 3 for Chloramphenicol). The final numberdefines the promoter that regulated the gene of interest (1 forPLtetO-1, 2 for PLlacO-1 and 3 for PA1lacO-1) and each of thesepromoters became activated by its corresponding inducer molecule (pLtetOcan be induced by tetracycline; pLlacO-1 and pA1lacO-1 can be induced byIPTG). Three base vectors, pZS*13S, pZA33S and pZE13S, were thendesigned and constructed to serve as “inducible” plasmid vectors.

In addition to the “inducible” promoters mentioned above, a set of“constitutive” promoters were sampled from the Registry(partsregistry.org). Each of these “constitutive” promoters was thenintroduced into the pZS*13S vector backbone to replace the pA1lacO-1inducible promoter via Sequence and Ligation Independent Cloning (SLIC)method described by Li & Eledge (Nature Methods 2007, 4:251-256). Ofthese sampled “constitutive” promoters (p100, p104, p105, p107, p108,p111, p115 & p119), experiments were carried out to establish an orderof promoter strength that was verified by protein expression levels. Forthe work discussed here, we employed both “inducible” and “constitutive”plasmid vectors, modified for the biobricks and SLIC insertions asdiscussed above. To further fine-tune protein expression levels of someoverly expressed proteins, ribosomal binding site (RBS) in betweenpromoter and gene coding sequence was modified accordingly using the RBScalculator (salis.psu.edu/software).

Mutagenesis Techniques—Error Prone-PCR

EpPCR (Pritchard et al., J Theor. Biol. 234:497-509 (2005)) introducesrandom point mutations by reducing the fidelity of DNA polymerase in PCRreactions by the addition of Mn²⁺ ions, by biasing dNTP concentrations,or by other conditional variations. The five step cloning process toconfine the mutagenesis to the target gene of interest involves: 1)error-prone PCR amplification of the gene of interest; 2) restrictionenzyme digestion; 3) gel purification of the desired DNA fragment; 4)ligation into a vector; 5) transformation of the gene variants into asuitable host and screening of the library for improved performance.This method can generate multiple mutations in a single genesimultaneously, which can be useful to screen a larger number ofpotential variants having a desired activity. A high number of mutantscan be generated by EpPCR, so a high-throughput screening assay or aselection method, for example, using robotics, is useful to identifythose with desirable characteristics.

Mutagenesis Techniques—Site Saturation Mutagenesis

In Site Saturation Mutagenesis, the starting materials are a supercoileddsDNA plasmid containing an insert and two primers which are degenerateat the desired site of mutations (Kretz et al., Methods Enzymol.388:3-11 (2004)). Primers carrying the mutation of interest, anneal tothe same sequence on opposite strands of DNA. The mutation is typicallyin the middle of the primer and flanked on each side by approximately 20nucleotides of correct sequence. The sequence in the primer is NNN orNNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A, C). Afterextension, DpnI is used to digest dam-methylated DNA to eliminate thewild-type template. This technique explores all possible amino acidsubstitutions at a given locus (that is, one codon). The techniquefacilitates the generation of all possible replacements at a single-sitewith no nonsense codons and results in equal to near-equalrepresentation of most possible alleles. This technique does not requireprior knowledge of the structure, mechanism, or domains of the targetenzyme. If followed by shuffling or Gene Reassembly, this technologycreates a diverse library of recombinants containing all possiblecombinations of single-site up-mutations. The usefulness of thistechnology combination has been demonstrated for the successfulevolution of over 50 different enzymes, and also for more than oneproperty in a given enzyme.

Combinatorial Mutants

MeDH Structure Model and Structures for Comparison

To design a library of mutations for improving the catalytic rates of2315, genes of several MeDHs as well as various Fe-dependent ADH geneswere aligned. The described structure/function relationships for theFe-dependent ADHs (40-47% sequence identity to MeDHs) were used toidentify regions of functional importance within the MeDH sequence. Analignment of the identified regions is presented in FIG. 4.

Blast search using the MeDH sequence against the PDB structure databasefound several structures of Fe-dependent alcohol dehydrogenases withsequence identities between 40 and 47%.

Similarities are spread out over the whole length of the protein. Giventhis similarity to known structures, the MeDH sequence was used togenerate a 3-dimensional model using the web-based iTasser structureprediction tool (Roy et al, Nature: Protocols, 5: 725-738 (2010)). Thefollowing three structures were specifically used for alignment andcomparison with the MeDH from Bacillus:

3OX4: ADH-2 from Zymomonas mobilis (sequence identity 47%)

1RRM: Lactaldehyde reductase from Escherichia coli (FucO, sequenceidentity 43%)

3BFJ: 1,3-PDO oxidoreductase from Klebsiella pneumoniae (DhaT, sequenceidentity 47%)

The Zymomonas enzyme was crystallized with bound cofactor and thestructure was well analyzed including the annotation of certain aminoacid residues for metal, cofactor and proposed substrate binding (EtOHmodeled into structure, Moon et al., J. Mol. Biol. 407, p 413-424,2011). Like the Lactaldehyde reductase (Montella et al., J. Bact. 187,p. 4957-4966, 2005) from E. coli, the Zymomonas ADH is a homodimer. Incontrast, the Klebsiella enzyme (Marcal et al., J. Bact. 191, p.1143-1151, 2009) was found to be a decamer with a structure thatresembles the MeDH appearance in electron microscopy studies.

Sequence comparison shows that all four coordination residues of theFe-dependent ADHs are conserved in the MeDH structure. Two of these fourresidues are in a histidine-rich sequence (residues 258-290) suggestedby Hektor et al. (J. Biol. Chem. 277, p. 46966-46973, 2002) as aputative metal binding site. As Fe and Zn share very similar bindingcharacteristics, the same amino acids responsible for the Fe-binding inthe ADHs may coordinate the Zn-atom of MeDH. From the alignment, thefollowing four amino acids are likely to constitute the metal bindingsite in MeDH (numbering transferred to Genomatica gene ID 2315): D193,H197, H262, H276

Amino acids considered important for cofactor binding in the ZymomonasADH are mostly conserved in the MeDH sequence. The respective residuesare listed below for Genomatica gene ID 2315. If the respective aminoacid differs, the Zymomonas ADH residue is noted in parentheses: D38,D70(N), G97, S98, T137, T138, T146, L148(F), L178

To cast a wide net, residues in a distance of 8 Å or less from the C1atom of propanediol were identified and are listed together with theannotated Zymomonas residues below. Residue numbers were transferred toGenomatica gene ID 2315 and the respective amino acids in the originalprotein are given in parentheses.

From Zymomonas ADH-2: L148(F149), V150(I151), P161(A162), F253(F254),L258(L259), H266(H267), D359(D360), V360(A361), C361(C362)

From E. coli FucO (≤8 Å distance): T141(T144), G142(A145), S143(A146),L148(N151), A149(Y152), H266(H267)

Mapping of the suggested mutagenesis sites onto the structure model ofMeDH shows that the residues selected as target sites line the entranceto the active site of the monomer. Positions 253, 258, 266, and 359 werefound to be strictly conserved, suggesting that they are more likely tobe essential for function and were therefore eliminated from the list ofresidues identified for mutations.

For the remaining five residues, amino acids for substitution wereselected based on their occurrence in related sequences. Only forposition 148 which was annotated with having a role in limiting thesubstrate size as well as positioning the nicotinamide ring in theactive site, a full panel of amino acids (NNK) is proposed. Narrowingdown the pool of substitutions in the other four positions made itpossible to include additional target sites while maintaining areasonable library size. The following three sites were added based ontheir proposed function and location in Fe-dependent ADHs: D70, T145,S147.

When comparing the variation for the respective positions in a sequencealignment it was noted that one of the residues is homolog to residue160 in an in house tested alcohol dehydrogenase. As a P160G mutationincreased the activity of this alcohol dehydrogenase, a glycine wasadded to the list of substitutions in the respective MeDH position. Thetable below summarizes the final list of targeted residues andsubstitutions (positions based on gene 2315A):

Position amino acids Variants 70 D, N 2 145 T, M 2 147 S, R 2 148 NNK 20150 V, I 2 161 P, A, V, G 4 360 V, A, G 3 361 C, N 2 Total 7680

TABLE 1 Amino acids mutations with respect to SEQ ID NO: 1, providinggreater than two fold activity when present as single mutations: Top InVitro Wild-type Hits Activity Amino Position (at least (Secondary InVivo Acid and and 3x in Screen; Formaldehyde Position SubstitutionPosition vitro + x = Top 16 (Average of Activity with with with 2x forinitial triplicate (fold over respect to respect respect incombinatorial Mutation assays) wild-type) 2315 to 2315 to 2315 vivo)library D60E 5.8 0.6 D60  60E 60 N87K 3.0 0.6 N87  87K 87 S99T 3.3 0.9S99  99T 99 A103V 3.1 1.9 A103 103V 103 V109Y 2.0 4.3 V109 109Y 109R115H 2.6 3.9 R115 115H 115 I116F 3.4 1.5 I116 116F 116 N117D 2.6 2.0N117 117D 117 G121D 2.7 2.0 G121 121D 121 x G121E 2.5 1.6 G121 121E 121G121L 2.7 1.3 G121 121L 121 G121M 2.6 1.8 G121 121M 121 G121R 2.8 1.7G121 121R 121 G121S 2.8 2.6 G121 121S 121 x G121T 2.7 G121 121T 121G121V 3.9 1.5 G121 121V 121 G121W 2.5 1.7 G121 121W 121 G121Y 3.1 1.5G121 121Y 121 V122P 2.5 2.3 V122 122P 122 N123L 2.7 1.8 N123 123L 123N123R 2.7 1.6 N123 123R 123 N123Y 3.0 1.8 N123 123Y 123 S124I 3.4 0.9S124 124I 124 S124L 2.4 1.2 S124 124L 124 V125C 2.6 3.2 V125 125C 125V125G 2.6 3.3 V125 125G 125 V125W 2.7 3.9 V125 125W 125 E126G 4.0 0.6E126 126G 126 K127C 2.6 3.9 K127 127C 127 K127R 2.5 3.3 K127 127R 127P128A 2.3 3.0 P128 128A 128 P128R 2.4 3.3 P128 128R 128 V129A 3.7 1.9V129 129A 129 V129M 4.7 1.4 V129 129M 129 X V129P 2.8 1.3 V129 129P 129V129S 3.0 1.5 V129 129S 129 V130F 2.1 1.4 V130 130F 130 V130Y 2.0 2.0V130 130Y 130 A134T 4.4 0.0 A134 134T 134 A149T 5.9 0.8 A149 149T 149V150A 3.0 1.2 V150 150A 150 K157N 3.6 0.8 K157 157N 157 V158E 2.6 4.3V158 158E 158 x V158H 2.2 2.8 V158 158H 158 V158K 2.0 2.6 V158 158K 158V158W 2.5 4.0 V158 158W 158 D164N 3.7 0.7 D164 164N 164 G270S 2.8 2.9G270 270S 270 x K345E 4.1 0.6 K345 345E 345 N355D 3.3 1.7 N355 355D 355x C361R 3.0 0.8 C361 361R 361 D38N 5.4 7.3 D38  38N 38 + P71I 7.6 2.5P71  71I 71 + P71V 6.8 3.5 P71  71V 71 + G107S 5.1 2.5 G107 107S 107 + xL108V 6.4 3.9 L108 108V 108 + L108W 7.4 4.8 L108 108W 108 + N117Q 3.34.3 N117 117Q 117 + N123D 3.0 2.0 N123 123D 123 + x N123I 3.1 2.5 N123123I 123 + X P128S 3.1 3.7 P128 128S 128 + V130I 3.4 2.3 V130 130I 130 +X S143T 3.8 2.4 S143 143T 143 + X T146N 3.4 2.0 T146 146N 146 + x A149L4.8 3.0 A149 149L 149 + A149M 4.6 2.7 A149 149M 149 + A149V 4.9 2.9 A149149V 149 + x I163Q 4.4 2.1 I163 163Q 163 + Q267H 6.3 4.3 Q267 267H 267 +X G270M 4.3 4.2 G270 270M 270 + G270Y 4.2 4.2 G270 270Y 270 + V360G 4.92.1 V360 360G 360 + x V360K 4.6 2.6 V360 360K 360 + V360R 4.6 3.4 V360360R 360 + x V360S 4.5 3.5 V360 360S 360 +

TABLE 2 Additional combination mutations with respect to SEQ ID NO: 1(generated from rationale design) In Vivo In Vitro Activity Activity(fald generation; (formal- average of dehyde Mutations triplicates)generation) D70N, L148G, P161G, V360A 6.2 2.92 D70N, L148G, V360A, C361N6.9 3.02 D70N, L148V, V150I, P161A, V360G 6.3 3.91 D70N, L148V, V360G6.6 3.95 D70N, P161A, V360A 6.2 3.93 D70N, P161V, V360G, C361N 5.6 3.95D70N, V150I, P161A, V360A 6.1 3.69 D70N, V150I, P161V, V360G, C361N 6.03.35 E48D, L148V, P161A, V360A 7.7 5.79 L148G, P161A, V360A, C361N 5.72.42 L148G, P161A, V360G 5.6 3.0 L148G, P161A, V360G, C361N 6.1 3.00L148G, P161G, V360A 7.4 3.0 L148G, P161G, V360G, C361N 6.8 2.50 L148G,V360A, C361N 6.3 3.80 L148G, V360G, C361N 6.6 3.67 L148I, P161G, V360G5.5 3.89 L148I, P161V, V360G 7.1 5.51 L148T, V150I, V360A 5.7 6.92L148T, V360G 5.6 6.26 L148V, P161A, V360A 7.5 5.7 L148V, V150I, P161A,V360A 6.4 5.7 L148V, V150I, P161A, V360A, C361N 6.1 1.97 L148V, V150I,P161A, V360G 8.2 5.27 L148V, V150I, P161A, V360G, C361N 6.6 4.23 L148V,V150I, P161A, V360G, C361N 5.9 3.80 L148V, V150I, P161G, V360A 6.6 5.14L148V, V150I, P161V, V360G, C361N 7.3 4.07 L148W, P161A, V360A, C361N5.7 1.06 N112K, S147R, P161A, V360A 9.7 5.40 P161A, Q217K, V360A, C361N6.7 4.15 P161A, V360A, C361N 8.0 3.58 P161A, V360G 7.4 4.8 P161V, E358G,V360G 6.0 4.96 P161V, V360A, C361N 9.7 5.2 P161V, V360G 6.4 4.0 P65Q,L148G, V150I, P161A, V360G, 5.5 2.32 C361N S147R, L148A, V150I, P161A,V360G 5.6 4.07 S147R, L148F, V150I, P161G, V360G 6.7 6.0 S147R, L148V,P161G, V360A 6.3 4.4 S147R, L148V, P161V, V360G 8.9 6.15 S147R, L148V,V150I, P161A, C361N 5.8 3.10 S147R, L148V, V150I, P161G, V360G 6.7 4.17S147R, P161A, V360A 6.3 5.53 S147R, P161A, V360A, C361N 6.6 3.6 S147R,P161A, V360G 5.7 5.22 S147R, P161V, V360G 10.0 5.84 S147R, P161V, V360G,C361N 5.7 2.11 S147R, V150I, P161V, V360A 5.7 5.13 S147R, V150I, V360A,C361N 5.6 4.66 T145M, L148I, V360G 8.0 3.48 V150I, I302V, V360G, C361N7.8 4.01 V150I, P161A, C361N 5.8 3.42 V150I, P161G, V360A, C361N 5.62.88 V150I, P161G, V360G 5.6 4.28 V150I, P161G, V360G, C361N 7.1 3.26V150I, P161V, C361N 5.7 3.9 V150I, P161V, K354R, V360A, C361N 8.8 4.24V150I, P161V, V360A, C361N 6.0 5.04 V150I, P161V, V360G, C361N 5.6 4.9V150I, V360A, C361N 6.4 4.43 V150I, V360G 6.8 6.79

TABLE 3 Additional combination mutations with respect to SEQ ID NO: 1(generated from epPCR) Mutations In Vitro Assay-Average of TriplicatesS11T, T74S, G269S, V344A 5.75 K84R, I163T 5.38 V122A, I163N 5.01 G107S,F333L 4.55 V129M, T152M, G343D 4.45 I63F, N355K 4.44 G107S, F333L 4.42E86K, S99T, A149V 4.41 N53I, V158E 4.38 N355I, K379M 4.30 H42Q, G107S4.08 Q120H, I163N 4.06 A149V, I323M 4.04 G107S, F333L 3.69 D164G, K181R3.68 A155V, R298H, N355D 3.66 N123D, E165G 3.65 I163F, L186M 3.65 G121A,T296S 3.63 I94V, S99P, N123I 3.62 E126V, V129M, V344G 3.60 Q120R, S143T3.58 G256C, A316V 3.56 P161Q, G312V 3.52 L226M, A300T, V360A 3.49 S337C,E350K, N355D, Q363K 3.43 D81G, V158E 3.42 I106L, N117Y, E126V 3.40G107S, G121D 3.36 V61A, V158E 3.31 N53I, V158E 3.28 N117Y, T190S 3.16S124R, I199V 3.13 K354M, C361R 2.97 A184T, C361R 2.86 E56K, Q267H 2.85S124R, E126G 2.79 T190A, N355K 2.77 P71T, F333L 2.75 G107S, F333L 2.74N123I, P336L 2.71

TABLE 4 Additional combination mutations with respect to SEQ ID NO: 1:in vivo Average Formaldehyde Mutation Secondary assay D38D/A149V 5.1 3.0D38N/V163V 6.6 9.9 D73D/L108V 6.8 3.9 G121R/P161S 4.4 2.8 G121R/P161S3.9 2.8 G121R/P161S 4.1 2.9 N112R/P161S 7.8 3.2

TABLE 5 Putative motifs and roles of the amino acid positions withrespect to SEQ ID NO: 1. Wild-type Sequence From To Length Rationale DAF38 40 3 NADH binding D 70 70 1 NADH binding G 95 95 1 Activation GS 9798 2 NADH & Activation TT 137 138 2 NADH binding TGS 141 143 3 NADHbinding TTSLAV 145 150 6 NADH & Substrate binding PVI 161 163 3Substrate & NADH (956 Gtp) L 178 178 1 NADH binding A 201 201 1 956 gTpF 253 253 1 Substrate binding L 258 258 1 Substrate binding H 266 266 1Substrate binding G 270 270 1 956 gtP DVC 359 361 3 Substrate binding 30Total

TABLE 6 In vivo assays showing formaldehyde (HCHO) production by variousNNOMO comprising a plasmid expressing a methanol dehydrogenase AccessionAccession Accession Accession number HCHO number HCHO number HCHO numberHCHO Experiment 1 (μM) Experiment 2 (μM) Experiment 3 (μM) Experiment 4(μM) EIJ77596.1 >50 EIJ77596.1 >50 EIJ77596.1 >50 EIJ77596.1 >50EIJ83020.1 >20 NP_00659.2 >50 NP_561852 >50 ZP_10241531.1 >50EIJ80770.1 >50 YP_004758576.1 >20 YP_002138168 >50 YP_005052855 >50ZP_10132907.1 >20 ZP_09352758.1 >50 YP_026233.1 >50 ZP_10132907.1 >50ZP_10132325.1 >20 ZP_10129817.1 >20 YP_001447544 >50 NP_617528 >50ZP_10131932.1 >50 YP_001139613.1 >20 Metalibrary >50 NP_617528 >50ZP_07048751.1 >50 NP_014555.1 >10 YP_359772 >50 ZP_08977641.1 >20YP_001699778.1 >50 WP_007139094.1 >10 ZP_01220157.1 >50 YP_237055 >20YP_004681552.1 >10 NP_343875.1 >1 ZP_07335453.1 >20 Empty vector <20ZP_10819291.1 <1 YP_006863258 >1 YP_001337153 >20 Empty vector 2.33NP_394301.1 >1 YP_694908 >20 ZP_10750164.1 >1 NP_717107 >20YP_023929.1 >1 AAC45651 >10 ZP_08977641.1 <1 ZP_11313277.1 >10ZP_10117398.1 <1 ZP_16224338.1 >10 YP_004108045.1 <1 YP_001113612 >10ZP_09753449.1 <1 YP_004860127 >10 Empty vector 0.17 YP_003310546 >10YP_001343716 >10 NP_717107 >10 YP_002434746 >10 Empty vector 0.11

TABLE 7 Wild-type enzymology Methanol Ethanol EtOH/MeOH k_(cat) K_(M)k_(cat)/K_(M) k_(cat) K_(M) k_(cat)/K_(M) k_(cat) k_(cat)/K_(M) (s⁻¹)(mM) (s⁻¹ mM⁻¹) (s⁻¹) (mM) (s⁻¹ mM⁻¹) (s⁻¹) (s⁻¹ mM⁻¹) MeDH B.methanolicus (2315A + 0.03 70 4.3 × 10⁻⁴ 0.16 209 7.7 × 10⁻⁴ 5.3 1.72317A) Human ADHB1 (2479B) 0.27 290 9.3 × 10⁻⁴ 2.85 1 2.85 11 3061Corynebacterium glutamicum 0.7 3 0.23 4.8 6.8 0.71 7 3 (2496B)Geobacillus 0.06 20 0.003 1.3 82 0.016 22 5 stearothermophilus (2480B)Saccharomyces cerevisiae Not available 340 17 20 Na Na (2497B)Flavobacterium frigidimaris Not available 27 0.17 158 Na Na (2499B)Escherichia coli (58) 0.047 2500 1.9 × 10⁻⁵ 1.5 115 0.013 32 699Clostridium perfringens (2430) 0.009 84 1.1 × 10⁻⁴ 0.73 33 0.022 91 232Geobacter bemijiensis (2449) 0.022 88 2.5 × 10⁻⁴ 0.95 72 0.013 43 53

TABLE 8 Wild type and variant enzymology k_(cat), K_(M), k_(cat)/K_(M),M⁻¹ Variant min⁻¹ mM min⁻¹ Wild-type 4.9 95 53 V360R 12 280 43 V360G 22130 170 S147R, L148F, V150I, P161G, V360G 4.4 370 12 P161V, V360A, C361N12 180 70 S147R, P161A, V360G 9.9 180 55 S147R, P161V, V360G 8.7 190 47N112K, S147R, P161A, V360A 4.2 210 20 A149V 7.2 410 18 Activity usingmethanol was determined. ^(a)Assays were performed at pH 7.6, 37° C. inthe presence of 2 mM NAD.

TABLE 9 Wild type and variant enzymology. k_(cat), K_(M), k_(cat)/K_(M),M⁻¹ Variant min⁻¹ mM min⁻¹ Wild-type 3.8 310 13 V360R 3.7 210 18 V360G3.2 280 12 S147R, L148F, V150I, P161G, V360G 5.3 240 23 P161V, V360A,C361N 52 110 500 S147R, P161A, V360G 15 190 76 S147R, P161V, V360G 15280 55 N112K, S147R, P161A, V360A 7.8 120 66 A149V 2.8 460 621,4-Butanediol-dependent steady-state kinetic parameters for wild-typeand variant methanol dehydrogenase.^(a) ^(a)Assays were performed at pH7.6, 37° C. in the presence of 2 mM NAD.

TABLE 10 Substitution templates AA/ NA SEQ % Tested ID AA Identity % #Activity NO: GenBankID GI No. Organism length (global) Similarity gap+++ 1/2 EIJ77596.1 387585261 Bacillus 382 100 100 0 methanolicus MGA3n.d 3/4 AAA22593.1 143175 Bacillus 381 97 99 0 methanolicus C1 + 5/6EIJ77618.1 387585284 Bacillus 383 93 96 0 methanolicus PB1 + 7/8EIJ78790.1 387586466 Bacillus 383 90 93 0 methanolicus PB1 +  9/10EIJ80770.1 387588449 Bacillus 385 62 79 1 methanolicus MGA3 ++ 11/12EIJ78397.1 387586073 Bacillus 385 61 78 1 methanolicus PB1 + 13/14EIJ83020.1 387590701 Bacillus 385 61 79 1 methanolicus MGA3 ++ 15/16EFI69743.1 298729190 Lysinibacillus 401 56 74 5 fusiformis + 17/18YP_004860127.1 347752562 Bacillus 386 56 76 1 coagulans 36D1 ++ 19/20YP_001699778.1 169829620 Lysinibacillus 402 54 73 5 sphaericus + 21/22ZP_11313277.1 410459529 Bacillus 386 54 73 1 azotoformans LMG 9581 n.d23/24 ZP_05587334.1 257139072 Burkholderia 390 54 70 2 thailandensisE264 + 25/26 YP_004681552.1 339322658 Cupriavidus 390 53 70 2 necatorN-1 n.d 27/28 AGF87161 451936849 uncultured 393 53 71 3 organism ++29/30 YP_002138168.1 197117741 Geobacter 387 52 71 1 bemidjiensis Bem ++31/32 YP_359772.1 78043360 Carboxydothermus 383 52 72 0 hydrogenoformansZ-2901 + 33/34 YP_001343716.1 152978087 Actinobacillus 385 51 71 1succinogenes 130Z + 35/36 ZP_16224338.1 421788018 Acinetobacter 390 5170 2 baumannii Naval-82 + 37/38 AAC45651.1 2393887 Clostridium 385 51 691 pasteurianum DSM 525 n.d 39/40 YP_007491369.1 452211255 Methanosarcina386 51 71 1 mazei Tuc01 n.d 41/42 YP_002434746 218885425 Desulfovibrio393 50 70 3 vulgaris str. ‘Miyazaki F’ ++ 43/44 YP_005052855 374301216Desulfovibrio 393 49 70 3 africanus str. Walvis Bay ++ 45/46 NP_561852.118309918 Clostridium 385 49 68 1 perfringens str. 13 ++ 47/48YP_001447544 156976638 Vibrio 382 49 69 0 campbellii ATCC BAA- 1116 +49/50 YP_001113612.1 134300116 Desulfotomaculum 388 49 70 2 reducensMI-1 n.d 51/52 YP_011618 46580810 Desulfovibrio 393 49 70 3 vulgarisstr. Hildenborough ++ 53/54 ZP_01220157.1 90412151 Photobacterium 382 4869 0 profundum 3TCK ++ 55/56 YP_003990729.1 312112413 Geobacillus sp.384 48 67 1 Y4.1MC1 + 57/58 ZP_07335453.1 303249216 Desulfovibrio 393 4869 3 fructosovorans JJ + 59/60 NP_717107 24373064 Shewanella 382 48 66 0oneidensis MR-1 + 61/62 YP_003310546.1 269122369 Sebaldella 384 48 68 1termitidis ATCC 33386 ++ 63/64 ZP_10241531.1 390456003 Paenibacillus 38447 67 1 peoriae KCTC 3763 + 65/66 YP_001337153.1 152972007 Klebsiella387 47 67 1 pneumoniae subsp. pneumoniae MGH 78578 ++ 67/68 YP_026233.149176377 Escherichia 383 46 64 0 coli + 69/70 YP_694908 110799824Clostridium 382 46 69 0 perfringens ATCC 13124 n.d 71/72 YP_725376.1113866887 Ralstonia 366 46 60 15 eutropha H16 n.d 73/74 YP_001663549167040564 Thermoanaerobacter 389 45 68 2 sp. X514 n.d 75/76 EKC54576406526935 human gut 384 37 55 3 metagenome n.d 77/78 YP_001126968.1138896515 Geobacillus 387 27 44 7 themodenitrificans NG80-2 Percentidentity is given based on global alignment to SEQ ID NO: 1.

TABLE 11 Template Polypeptides Sequences of Template Polypeptidesindicating exemplary substitutions, their positions and theircorresponding positions in other template sequences: SEQ ID NO: GI No.Protein Sequence 1 387585261 MTTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVT DAFLHSTGLSEEVAKNIREAGV D VAIFPKAQPD P ADTQVHEGVDVFKQE N CDSLVSIGGGS S HDTA KAI GLV AANGG RIN D YQ GVNSVEKPVV PVVAITTTAGTG S ET T SL AV ITDSAR KVKMPV ID EKITPTVAIVDPELMVKKPAGLTIATGMDALSHAIEAYVAKGATPVTDAFAIQAMKLINEYLPKAVANGEDIEAREKMAYAQYMAGVAFNNGGLGLVHSISH Q VG G VYKLQHGICNSVNMPHVCAFNLIAKTERFAHIAELLGENVAGLSTAAAAERAIVALERINKSFGIPSGYAEMGV K EEDIELLAK N AYEDVC TQSNPRVPTVQDIAQIIKNAM 3 143175 MTNFFIPPASVIGRGAVKEVGTRLKQIGAKKALIVT DAFLHSTGLSEEVAKNIREAGL D V AIFPKAQPD P ADTQVHEGVDVFKQE N CDALVSIGGGS SHDT A KAI GLV AANGG RIN DY Q GVNSVEKPVV PVVAITTTAGTG S ET T SL AV ITDSARKV KMPV ID EKITPTVAIVDPELMVKKPAGLTIATGMDALSHAIEAYVAKGATPVTDAFAIQAMKLINEYLPKAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVHSISH Q VG G VYKLQHGICNSVNMPHVCAFNLIAKTERFAHIAELLGENVSGLSTAAAAERAIVALERYNKNFGIPSGYAEMGV K EEDIELLAK N AFED VCTQSNPRVATVQDIAQIIKNAL 5 387585284 MTQRNFFIPPASVIGRGAVKEVGTRLKQIGATKALIVTD AFLHGTGLSEEVAKNIREAGL D AVIFPKAQPD P ADTQVHEGVDIFKQE K CDALVSIGGGS SHDT A KAI GLV AANGG RIN DYQ GVNSVEKPVV PVVAITTTAGTG S ET T SL AV ITDSARKV KMPV ID EKITPTVAIVDPELMVKKPAGLTIATGMDALSHAIEAYVAKRATPVTDAFAIQAMKLINEYLPRAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVHSISH Q VG G VYKLQHGICNSVNMPHVCQFNLIARTERFAHIAELLGENVSGLSTASAAERAIVALQRYNKNFGIPSGYAEMGV K EEDIELL AN NAYQD VC TLDNPRVPTVQDIAQIIKNAL 7 387586466MTKTKFFIPSSTVFGRGAVKEVGARLKAIGATKALIVT D AFLHSTGLSEEVAKNIREAGL DVVIFPKAQPD P ADTQVHEGVEVFKQE K CDALVSIGGGS S HDT A KGI GLV AANGG RIN DYQ GVNSVEKQVV PQIAITTTAGTG S ET T SL AV ITDSAR KV KMPV ID EKITPTVAIVDPELMVKKPAGLTIATGMDALSHAIEAYVAKRATPVTDAFAIQAMKLINEYLPKAVANGEDIEAREAMAYAQYMAGVAFNNGGLGLVHSISH Q VG G VYKLQHGICNSVVMPHVCQFNLIARTERFAHIAELLGENVSGLSTASAAERTIAALERYNRNFGIPSGYKAMGV K EEDIELLAN N AMQDVC TLDNPRVPTVQDIQQIIKNAL 9 387588449MKNTQSAFYMPSVNLFGAGSVNEVGTRLAGLGVKKALLVT D AGLHSLGLSEKIAGIIRE AGV EVAIFPKAEPN P TDKNVAEGLEAYNAE N CDSIVTLGGGS S HDA G KAI ALV AANGG TIH DYEGVDVSKKPMV PLIAINTTAGTG S EL T KF TI ITDTER KV KMAI VD KHVTPTLSINDPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIKIISKYLPRAVANGKDIEAREQMAFAQSLAGMAFNNAGLGYVHAIAH Q LG G FYNFPHGVCNAILLPHVCRFNLISKVERYAEIAAFLGENVDGLSTYEAAEKAIKAIERMARDLNIPKGFKELGA K EEDIETLAK N AMNDAC ALTNPRKPKLEEVIQIIKNAM 11 387586073MTNTQSIFYIPSVNLFGPGSVNEVGTRLAGLGVKKALLVT D AGLHGLGLSEKIASIIREAG V EVLIFPKAEPN P TDKNVAEGLEVYNAE N CDSIVTLGGGS S HDA G KGI ALV AANGG TIY DYEGVDKSKKPMV PLIAINTTAGTG S EL T RF TI ITDTER KV KMAI VD KHVTPTLSINDPELMVGMPPSLTAATGLDALTHAIEAYVSTAATPITDALAIQAIKIISKYLPRAFANGKDMEAREQMAFAQSLAGMAFNNASLGYVHAIAH Q FG G FYNFPHGVCNAILLPHVCRFNLISKVERFAEIAALLGENVAGLSTREAAEKGIKAIERMAKDLNIPRGFKELGA K EEDIVTLAE N A MKD ATALTNPRKPKLEEVIQIIKNAM 13 387590701MTNTQSAFFMPSVNLFGAGSVNEVGTRLADLGVKKALLVT D AGLHGLGLSEKISSIIRA AGV EVSIFPKAEPN P TDKNVAEGLEAYNAE N CDSIVTLGGGS S HDA G KAI ALV AANGG KIH DYEGVDVSKEPMV PLIAINTTAGTG S EL T KF TI ITDTER KV KMAI VD KHVTPTLSINDPELMVGMPPSLTAATGLDALTHAIEAYVSTGATPITDALAIQAIKIISKYLPRAVANGKDIEAREQMAFAQSLAGMAFNNAGLGYVHAIAH Q LG G FYNFPHGVCNAVLLPYVCRFNLISKVERYAEIAAFLGENVDGLSTYDAAEKAIKAIERMAKDLNIPKGFKELGA K EEDIETLAK N AMKDAC ALTNPRKPKLEEVIQIIKNAM 15 298729190MSDVLKQFVMPKTNLFGPGAIQEVGKRLNDLEVKKTLIVT D EGLHKLGLSEQIANIITAA GI DVAIFPKAEPN P TDQNIEDGISVYHAE N CDSIVSLGGGS A HDA A KGI GLI ASNGG RIH DYEGVDKSQNPLV PLIAINTTAGTA S EM T RF TI ITDTAR KV KMAI VD KHVTPLLSINDPELMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQLIPEYLPRAYANGADIEAREQMVYAQFLAGMAFNNASLGYVHAIAH Q LG G FYNLPHGVCNAILLPHVCRFNVTARTERFARIAELLGENVEGLSKRDAAEKAITAIEKLSQDLNIPSGFRELGA K DEDIEILAK N ALL D VCAETNPRKATLEDIKQIITNAMGPIVKKEESLEAVALS 17 347752562MLTGLRTDFQMPSVNLFGQGTAEEIGNRLKNLGCRRPLIVT D EGLHQLGYSEKIAAYIKE AGL EVAIYPKAEPN P TDKNVEDGLKTYHEE N CDSIVSLGGGS A HDC A KGI GLV AANGG KIH DYEGLDRSEKPMV PLVAINTTAGTA S EM T KF TI ITDTSR KV KMAI VD KHVTPVLSINDPLLMVGMPPSLTAATGLDALTHAVEAYVSTAATPVTDACAIKAIQIIPQYLPKAVANGNDMEAREQMVYAQYLAGMAFNNASLGYVHAIAH Q FG G FYNLPHGVCNAILLPHVCRFNLIARKERFAEIAVALGEKTDSLSVDEAAEKAITAIERLAAQLNIPKGFKELGA K EEDIEIL AQ HAMQD AC AATNPRKPTQKEVEAIIKAAM 19 169829620MSDVLKQFVMPKKNLFGPGAIQEVGKHLNDLEVKKTLIVT D EGLHKLGLSEQIANIITAA GI DVAIFPKAEPN P TDQNIEDGIADYHAE S CDSIVSLGGGS A HDA A KGI GLI ASNGG RIQ DYEGVDKSQNPLV PLIAINTTAGTA S EM T RF TI ITDTAR KV KMAI VD KHVTPLLSINDSELMIGLPPALTAATGVDALTHAIESFVSTNATPITDACAEKVLQLVPEFLPRAYANGADLEAREQMVYAQFLAGMAFNNASLGYVHAIAH Q LG G YYNLPHGVCNAILLPHVCRFNVTARTERFARIAELLGENVTGLSKRDAAEKAISAIEKLSKDLNIPSGFRELGA K DEDIEILAK N A MLDVC AETNPRKATLDDIKQIITNAMGPIVKKEESLEAVAALS 21 410459529MANQKVYGFFMPTVNLMGVGAVNEAGPRIKALGCNKSLLVT D KGLSKMGVAEEIANI IGQAGV EVSIFDGAEPN P TDLNVEAGLKQYREL G CDSIISLGGGS S HDC A KGI GLV ASNG G TIHDYE GVDMSKEPMI PLVAINTTAGTA S EM T RF CI ITDTSR KI KMAI VD KHTTPLISINDPILTVKMPAGLTAATGMDALTHAIEAYVSTDATPITDACALQTIRLVSQNLRAAVANGEDIDARNNMCYAQFLGGMAFNNASLGYVHAIAH Q LG G FYNLPHGVCNAVLLPHVERFNLIAKPERFVDIAIALGENVSGLPTRAAAEIALTAIETLAKDVGIPGSLTELGV K EEDIPLLA E NAMRD AC SFTNPRKATLDDVQGMIRAAL 23 257139072MSYLNIAQRTDSFFIPCVTLIGPGCARETGVRAKSLGAKKALIVT D AGLHKMGLSEIVAG HIRDAGL QAVIFAGAEPN P TDVNVHDGVERFQRE G CDFIVSLGGGS S HDC A KGI GLV T AGGG HIRDYE GIDKSTVPMT PLISINTTAGTA A EM T RF CI ITNSSN HV KMAI VD WRCTPLIAIDDPCLMVAMPPALTAATGMDALTHAVEAYVSTAATPITDACAEKAIALIGEWLPKAVANGESMEARAAMCYAQYLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVCEFNLIAAPERFATIASLLGVNTAGSSTVDAARAGHAAIPRLSASIGIPAGLAALGV RVEDHEVMAS N AQKD AC MLTNPRKATLAQVIAIFAAAM 25 339322658MTHLNIANRVDSFFIPCVTLFGPGCARETGARARSLGARKALIVT D AGLHKMGLSEVVA GHIREAGL QAVIFPGAEPN P TDVNVHDGVKLFERE E CDFIVSLGGGS S HDC A KGI GLV T AGGG HIRDYE GIDKSTVPMT PLISINTTAGTA A EM T RF CI ITNSSN HV KMAI VD WRCTPLIAIDDPSLMVAMPPALTAATGMDALTHAIEAYVSTAATPITDACAEKAIVLIAEWLPKAVANGDSMEARAAMCYAQYLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVSEFNLIAAPERYARIAELLGENIGGLSAHDAAKAAVSAIRTLSTSIGIPAGLAGLGV K ADDHEVMAS N AQKD AC MLTNPRKATLAQVMAIFAAAM 27 451936849MSLVNYLQLADRTDGFFIPSVTLVGPGCVKEVGPRAKMLGAKRALIVT D AGLHKMGLSQEIADLLRSEGI D SVIFAGAEPN P TDINVHDGVKVYQKE K CDFIVSLGGGS S HDC A KGI GLV TAGGG HIR DYE GVDKSKVPMT PLIAINTTAGTA S EM T RF CI ITNTDT HV KMAI VDW RCTPLVAIDDPRLMVKMPPALTAATGMDALTHAVEAYVSTAATPITDTCAEKAIELIGQWLPKAVANGDWMEARAAMCYAQYLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVCQFNLIAATERYARIAALLGVDTSGMETREAALAAIAAIKELSSSIGIPRGL SELGV KAADHKVMAE N AQKD AC MLTNPRKATLEQVIGIFEAAM 29 197117741MALGEQTYGFYIPTVSLMGIGSAKETGGQIKALGASKALIVT D KGLSAMGVADKIKSQV EEAGV SAVIFDGAEPN P TDINVHDGVKVYQDN G CDAIISLGGGS S HDC A KGI GMV IGN GG HIRDLE GVNKTTKPMP AFVAINTTAGTA S EM T RF CI ITNTDT HV KMAI VD WRCTPNVAINDPLLMVGKPAALTAATGMDALTHAVEAYVSTIATPITDACAIKAIELIAEFLSKAVANGEDLEARDKMAYAEYLAGMAFNNASLGYVHSMAH Q LG G FYNLPHGVCNAILLPAVSQYNLIACPKRFADIAKALGENIDGLSVTEAGQKAIDRIRTLSASIGIPTGLKALNV K EAD LTIMAEN AKKD AC QFTNPRKATLEQVVQIFKDAM 31 78043360MKTYRFYMPPVSLMGIGCLKEAGEEIKKLGFKKALIVT D KVLVKIGLVNKLTEILDNEGI EYVIFDETKPN P TVKNVEDGLKMLKEN N CDFLISFGGGS P HDC A KGI GLV ATNGG SIK DYE GVNKSAKPML PLVAVNTTAGTA S EM T RF SI ITDEDR HV KMAI VD WHVTPIMAVNDPELMVEMPKALTAATGMDALTHAIEAYVSIDATPVTDAAALKAIELIFKYLKRAVENGKDIEARDKMAYAEYLAGVAFNNAGLGYVHAMAH Q LG G FYDLPHGVCNAVLLPHVQAYNLQVVPERFIDIAKAMGINVENLTAKEAGEKVLEAIKNLSREIGIPSGLKELGV K EEDLKTLAE N ALKDAC GFTNPKQASLDDIIRIFKEAM 33 152978087MSTYYFLPTRNVFGENAVEEVGTLMKSLGGNNPLIVT D AFLAKNGMADQLAAVLSNA GL KPVIFGGAEPN P TDKNVEEGIVFYNEH G CDSIISLGGGS S HDC A KGI GLI ASNGG RIQ DYEGVDRSHNAMV PLMAVNTTAGTA S EI T RF CI ITDTAR KV KMAI VD WRITPQIAVNDPLLMKGMPPSLTAATGMDALTHAIEAYVSTAANPLTDAAALMAITMIQQYLPKAVANGDYMKARDKMAYAQYLAGIAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPYVEEFNLIGNLNRFRDIAKAMGENIDGLCTDDAALKAIGAIRRLSKQVGIPANLQLLGV K PEDF DVMAE NAMKD VC MLTNPRKATKQQVIEIFQRAYDGD 35 421788018MAFKNIADQTNGFYIPCVSLFGPGCAKEIGTKAQNLGAKKALIVT D EGLFKFGVADLIAS YLTEAGV ASHIFPGAEPN P TDINVHNGVNAYNEN G CDFIVSLGGGS S HDC A KGI GLV TA GGG HIRDYE GIDKSKVPMT PLIAVNTTAGTA S EM T RF CI ITNTDT HV KMAI VD WRCTPLIAIDDPKLMIAKPAGLTAATGMDALTHAVEAYVSTAANPITDACAEKAITMISQWLQPAVANGENIEARDAMSYAQYLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVCEFNLIACPDRYAKIAELMGVNTHGLTVTEAAYAAIDAIRKLSSLIGIPSGLTELGV K TEDLAVMAE N AQKD AC MLTNPRKANHAQVVEIFKAAL 37 2393887MRMYDFLAPNVNFMGAGAIKLVGERCKILGGKKALIVT D KFLRNMEDGAVAQTVKYIK EAGI DVAFYDDVEPN P KDTNVRDGLKVYRKE N CDLIVTVGGGS S HDC G KGI GIA ATHE G DLYDYA GIETLTNPLP PIVAVNTTAGTG S EV T RH CV ITNTKT KI KFVI VS WRNLPLVSINDPILMIKKPAGLTAATGMDALTHAIESYVSKDANPVTDALAIQAIKLIANNLRQAVALGENLEARENMAYASLLAGMAFNNANLGYVHAMAH Q LG G LYDMAHGVANAMLLPHVERYNLISNPKKFADIAEFMGENIEGLSVMEAAEKAIDAMFRLSKDVGIPASLKEMGV N EG DFEYMAK MALKD GN AFSNPRKGNEKDIVKIFREAF 39 452211255MIEKMTYTYLNPKIALMGPGCVNGIGTHAKDLGGTKALIVS G KSRHGKELAADIRRILER AGI EAAIFPGADPN P TDTSVMEGADIYRKE N CNMIVAVGGGS P MDC A KAI GIV VYNG G RINDYE GVGKVTRGIP PLITVNTTAGTA S EM T SF TI ITDTER HI KMAI VD PRITPDVAVNDPELMVSMPPALTAATGMDALTHAVEAYVSTMATPTTDAAAIKAIELISKYLPEAVLHGEDIRARDMMAHAEYLAGIAFNNASLGYVHSMAH Q LG G FYDLPHGVCNAILLPYVEMYNKQVCPERFADIAKAMGEKVEGLSPEEAADKAIEAIKKLAAEIGIPSGLKELGA R EEDLE LLAE NAMQD VC RLTNPRELSKEDIIEIYRKAL 41 218885425MAVQEQVYGFFIPSVTLIGIGASKAIPEKIKALGGSKPLIVT D MGIVKAGILKQITDLLDAA KM AYSVYDETIPN P TDDNVHKGVEVYKKN K CDSLITLGGGS S HDC G KGI GLV IANGG KI HDFE GVDKSFKPMP PYVAVNTTAGTA S EM T RF CI ITDTSR KV KMAI VD WRVTPSIALDDPLLMMGMPPALTAATGMDALTHAVEAYVSTIATPMTDACAEQAITLIATFLRRAVANGRDIEARERMCFAQYLAGMAFNNASLGHVHAMAH Q LG G FYDLPHGECNAILLPHVSQFNLIAKLDRFARIAELMGENISGLSVRDAAEKAICAIKRLSADVGIPAGLVALGKRYGK DV KAKDIAIMTK N AQKD AC GLTNPRCPTDADVAAIYEAAM 43 374301216MAVREQVYGFFIPSVTLIGIGASKEIPNKIRDLGGKKPLIVT D QGIVKAGILKMITDHMDK AGM QYSVYDKTIPN P TDNNVAEGVEVYKKE G CDSLITLGGGS S HDC G KGV GLV VSNG G KIHDYE GVDKSTKPLP PYVAVNTTAGTA S EM T RF CI ITDTSR KV KMAl VD WRVTPGIALDDPLLMVGMPPALTAATGMDALTHAVEAYVSTIATPMTDACAEKAISLIFTFLRRATANGQDIEAREGMCFAQYLAGMAFNNASLGHVHAMAH Q LG G FYDLPHGECNAILLPHVEKYNLIAKVERFGKMAEIMGENIQGMSPRAAAEKCLDAIRQLSQDVGIPSGLIELGKRY GKNV KKEDIDTMTG N AQKD AC GFTNPRCPSDKDVKAIYEAAL 45 18309918MRMYDYLVPSVNFMGANSISVVGERCKILGGKKALIVT D KFLRGLKGGAVELTEKYLKE AGI EVAYYDGVEPN P KDTNVKDGLKIFQDE N CDMIVTVGGGS S HDC G KGI GIA ATHEG DLY DYAGIETLTNPLP PIVAVNTTAGTA S EV RH CV ITNTKT KV KFVI VS WRNLPLVSINDPMLMVGKPAGLTAATGMDALTHAVEAYVSKDANPVTDAAAIQAIKLISSNLRQAVALGENLVARENMAYGSLLAGMAFNNANLGYVHAMAH Q LG G LYDMPHGVANAMLLPHVCKYNLISNPQKFADIAEFMGENIEGLSVMDAAQKAIDAMFRLSTDIGIPAKLRDMGV K EEDFGYMAE MALKD GN AFSNPRKGNERDIVEIFKAAF 47 156976638MTSAFFIPTVNLMGAGCLKDATDSIQSQGFKKGLIVT D KILNQIGVVKQVQDLLAERDV ETVVFDGTQPN P TISNVNDGLALLTDN E CDFVISLGGGS P HDC A KGI ALV ASNGG KIA DYE GVDQSAKPMM PLIAINTTAGTA S EM T RF CI ITDEER HI KMAI VD KHTTPLISVNDPELMLAKPASLTAATGMDALTHAIEAYVSIAATPITDAVAIKAIELIQAYLRTAVKNGEDLEAREQMAYAQFMAGMAFNNASLGYVHAMAH Q LG G FYDLPHGVCNAILLPHVQRYNAQVCPERLRDVAKAMGVNVEDMSAEAGAAAAIDAIVTLAKDVGIPAGIKELGA K LEDIPTL AD N ALKDAC GFTNPKQATHEEISKIFEEAM 49 134300116MTVGEQVFGYFIPTVNLMGVGAHKEIPDQVKVLGGSNVLIVT D AFLGRPGGMADDIK GMLEAENI KVTIYAGAEPN P TDVNVHDGLKVYQ E CGADMILSLGGGS S HDC A KGI GIV ATNGG NIR DYEGINKSSKAMP PFIAVNTTAGTA S EM T RF CI ITNTSN HV KMAI VD WRCTPNIAINDPLLMAGMPPALTAATGMDALTHAIEAYVSVAATPVTDSAALMAIKLISQYLRAAVANGENMEARDKMAYAEFLGGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVEAFNLIACPERFVDIAVAMGENVEGLSVRDAADKALSAIRKLSADVGIPAGLTE LGV KEEDLKTMAE N AMKD AC ALTNPRKATLNDIVGIYKTAL 51 46580810MAVQEQVYGFFIPRVTLIGIGASKAIPEKIKALGGSKPLIVT D MGIVKAGILKQITDLLDAA KM AYSVYDETIPN P TDDNVHKGVDVYKKN K CDSLITLGGGS S HDC G KGI GLV VANGG K IHDFE GVDKSTQRMP PYLAVNTTAGTA S EM T RF CI ITDTSR KV KMAI VD WRVTPNIALDDPLLMLGMPPALTAATGMDALTHAVEAYVSTIATPMTDACAEQAITLIATFLRRAVANGQDLEARERMCFAQYLAGMAFNNASLGHVHAMAH Q LG G FYDLPHGECNAILLPHVSKFNLIAKLDRYARIAQLMGENIAGLSTREAAERAISAIKCLSTDVGIPAGLVALGKRYGKDVKAADIAIMTK N AQKD AC GLTNPRCPTDADVAAIYEAAL 53 90412151MSSAFFIPSVNLMGAGCLTEAADAVKAHGFKKALIVT D KVLNQIGVVKQVVDLLAERN V EAVVFDGTQPN P TMGNVEAGLALLKAN E CDFVISLGGGS P HDC A KGI ALV ASNGG SI SDYE GVDVSAKPQL PLVAINTTAGTA S EM T RF CI ITDEAR HI KMAI VD KNTTPLMSVNDPELMLAKPASLTAATGMDALTHAIEAYVSTAATPITDAVAIKAMELIQAHLRTAVNDGQNLEAREQMAYAQFMAGMAFNNASLGYVHAMAH Q LG G FYDLPHGVCNAVLLPHVQRYNAKVCPERLRDVAKAMGVNVEAMTADQGADAALEAIQVLSKDVGIPAGLKDLGA K NEDISILAD NALKD AC GFTNPKQATHEEISEIFAAAM 55 312112413MSNAHVFYVPSTNLMGRGCLAKVGPFIKEFGFKKALVVT D KFLHKSGIAGKVLAVLDEI GV NYVVYDDVKPN P TTKNVYAGADLFKKN E CDFLVSVGGGS P QDT A KAI GLY VTNGG DIR DYEGVNKTKNKSV PIVAVNTTAGTS S EF T IN YV ITDEER NV KMVM VD KNSLVTISVNDPELMVDKPAALTAATGMDALTHAIEAVVTPGSYTVTDATALAAIEIIFNYLPRAVKNGHDIEAREQMAYAMFLVGIAFNNAGLGMVHAMAH Q LG G MYDLPHGVCNAMLLPIVERENAKRDPRKFRAIAKAAGIDVTGKTDEQCAEEVIEAIKALSREIGIPSKLSELGV D EVDL EKLAN NALKD AC APGNPFQPTKEEVISMFKEIL 57 303249216MAVREQVYGFFIPSVTLIGIGAAKQIPEKIKALGGTKPLIVT D KGVVKVGVCKMITDLLDA AGM KYHIYDETIPN P TDENVHKGVEVYKKE G CDSLITLGGGS S HDC G KGI GLV ISNGG KI HDYE GVDKSSKPFM PYLAVNTTAGTA S EM T RF CI ITDLSR HV KMAI VD WRVTPHIAIDDPVLMVGMPPALTASTGMDALTHAVEAFVSTIANPMTDACAIEAIKLIFKYLRKAVANGQDMEAREGMCFAEYLAGMAFNNASLGHVHAMAH Q LG G FYDLPHGECNAILLPHVESYNLIAKVEKFAEMAKIMGENIEGMAPRDAAELCLKAIRQLSVDVGIPAGLVELGKRY GKDV KAADIPTMTG N AQKD AC GLTNPRCPTDKDVAAIYTAAL 59 24373064MAAKFFIPSVNVLGKGAVDDAIGDIKTLGFKRALIVT D KPLVNIGLVGEVAEKLGQNGI TSTVFDGVQPN P TVGNVEAGLALLKAN Q CDFVISLGGGS P HDC A KGI ALV ATNGG SIK DYE GLDKSTKPQL PLVAINTTAGTA S EM T RF CI ITDEAR HI KMAI VD KHTTPILSVNDPELMLKKPASLTAATGMDALTHAVEAYVSIAANPITDACAIKAIELIQGNLVNAVKQGQDIEAREQMAYAQFLAGMAFNNASLGYVHAMAH Q LG G FYDLPHGVCNALLLPHVQEYNAKVVPHRLKDIAKAMGVDVAKMTDEQGAAAAITAIKTLSVAVNIPENLTLLGV K AEDIPT LAD N ALKDAC GFTNPKQATHAEICQIFTNAL 61 269122369MKVSRRIYWPAVTLIGPGCVKEIGGDIKDLGLKKALVVT D NVLVKIGVVKKVTDVLDESG I NYVVVDDIQPN P TMKNIHDGLNTYKSE N CDFVISIGGGS P QDA G KAI GLL ATNGG EIK DYEGINMSKHKSV PIIAINTTAGTA S EV T IN YV ITNEDT HI KMVM VD KNCLASIAVSDPELMTGKPADLTAATGMDALTHAIEAYVSTGAYELTDVLALEAVKLIGESLEDAVKDGNNIEARSKMAYASYIAGMSFNNAGLGYVHSMAH Q LG G FYNLPHGVCNAILLPHVEKFNSANTGDKLRKVAEILGENVEGLSVEEANAKAIEAIMKLSERVGIPKGLKELGV K EEDFKVMA E N ALKDVC AGTNPREVTLEDTIALYKEAL 63 390456003MTGTSKFMMPGMSLMGSGALADAGTEIGKLGYTNALIVT D KPLVDIGIVKKVTSVLESI NV KSVVYSGTQPN P TVTNVNEGLELLSQS K CDFIISLGGGS P HDC A KGI ALL ASNGG QI GDYE GVDKSTKPSF PLIAINTTAGTA S EM T MF CI ITDEER HI KMAI VD NHTTPLIAVNDPDLMMAMPKSLTAATGMDALTHSIEAYVSTNATPITDACAIKAIELIRDNLARAVDDGNDVEARSQMAYAEFLAGMAFNNAGLGFVHAMAH Q LG G FYNLPHGVCNAILLPHVERYNAKASAERLTDIARALGENTDGVTPEQGANLALQAIEKLAKRVNIPSGLEELGV K REDFT VLAA NALKD AC GVTNPVQPTQQEVIAIFEQAM 65 152972007MSYRMFDYLVPNVNFFGPNAISVVGERCQLLGGKKALLVT D KGLRAIKDGAVDKTLHY LREAGI EVAIFDGVEPN P KDTNVRDGLAVFRRE Q CDIIVTVGGGS P HDC G KGI GIA ATH EG DLYQYA GIETLTNPLP PIVAVNTTAGTA S EV T RH CV LTNTET KV KFVI VS WRNLPSVSINDPLLMIGKPAALTAATGMDALTHAVEAYISKDANPVTDAAAMQAIRLIARNLRQAVALGSNLQARENMAYASLLAGMAFNNANLGYVHAMAH Q LG G LYDMPHGVANAVLLPHVARYNLIANPEKFADIAELMGENITGLSTLDAAEKAIAAITRLSMDIGIPQHLRDLGV K E ADFPYMAEM ALKD GN AFSNPRKGNEQEIAAIFRQAF 67 49176377MAASTFFIPSVNVIGADSLTDAMNMMADYGFTRTLIVT D NMLTKLGMAGDVQKALE ERNI FSVIYDGTQPN P TTENVAAGLKLLKEN N CDSVISLGGGS P HDC A KGI ALV AANGG DIR DYEGVDRSAKPQL PMIAINTTAGTA S EM T RF CI ITDEAR HI KMAI VD KHVTPLLSVNDSSLMIGMPKSLTAATGMDALTHAIEAYVSIAATPITDACALKAVTMIAENLPLAVEDGSNAKAREAMAYAQFLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAVLLPHVQVFNSKVAAARLRDCAAAMGVNVTGKNDAEGAEACINAIRELAKKVDIPAGLRDLNV K EE DFAVLAT NALKD AC GFTNPIQATHEEIVAIYRAAM 69 110799824MSYKFFMPAISLMGADCLKDAGDQVGELGFKKALIVT D KVLGQIGIVKKVTDVLDNKNI EYAIYDETKPN P TVKNVNDGLALLKEK E CDFVISLGGGS A HDCAKGI ALL ATNGG EIK DY EGVDKSKKPQL PMVGINTTAGTG S EM T LF AI ITDEER HI KMAL VD KHLTPIIAVNDPILMLAMPKSLTAATGMDALTHAIEAYVSTAATPITDACAEKAIELISNYLVNAVENGQDVEARDMMAYAEYLAGMAFNNASLGYVHAMAH Q LG G FYNLPHGVCNAILLPHVQEYNKSTSASRLAKIAKIMGGNIEGLTDEQGADLCIDMIKSLSQTIGIPEGLGVLGV K ESDFETLAT N ALNDAC SLTNPRKGNLEEVIAIFKKAM 71 113866887MRARPARAPKRKAQERPSSSRMPACTRWGYPKPSRGTSARQGF R PLIFPGAEPN P TDVNVHDGVKLFEQE G CDFIVSLGGGS S HDC A KGI GLV TAGGG HIR DYE GIDKSTVPMT PLISINTTAGTA A EM T RF CI ITNSSN HV KMAI VD WRCTPLIAIDDPRLMVAMPPALTAATGMDALTHAVEAYVSTAATPITDACAEKAIALIGEWLPKAVANGNSLEARAAMCYAQYLAGMAFNNASLGYVHAMAH Q LG GL YNLPHGVCNAILLPHVSEFNLIAAPERFAKIAELLGENVASLSTSDAAKAAISAIRALAASIGIPAGLASLGV K AEDHEVMAH N AQKD AC MLTNPRRATTAQVIAIFAAAM 73 167040564 MKIFKFHMPPINLIGVGCLKDVGREIKKLGFKKGIIVT DKVLVRAGLVNNVISVLEEEGI E Y VVFDETKPN P TIKNVTNGLKLLIEN K CDFIISCGGGS AHDC A KGI GLI AKEKN FID EVE RL DKVK CGGWNS ALLL PLVAINTTAGTG S EV T KFAI ITDEEK RI KMPI VD WRITPLIAVNDPLLMIGMPKSLTAASGMDALTHAIEAYISIDANPFTDALALKAIEIIFNYLKRAVENGNDIEAREKMAYAEFLAGIAFNNAGLGYVHAMAH Q LG G FYDLPHGVCNAVLLPHVLEYNLEAVQNKLIYIAKAMGIDVDKLTTKEIGGKIIESINQLSQEIGIPSRLKELGV K EEDIKELSQ N AL KDVC GFTNPKKATLEDIINIFKSAM 75 406526935MGNRIILNGTSYFGRGARENVITELRNRNFTKALVVT D KNLLDAHVTNLVTDVLDKNDFSYQIYSDIKPN P TTLNVQEGVTFCRNSKADVIIAVGGGS A IDT A KAI SII MTNPE HFD VISLD GAVETKNAGM PIIALPTTAGTA A EV T IN YV ITNPVG PK KMVC VD PHDIPIVAIIDQDLMEKMPKSLAASTGMDALTHAMEGYTTKAAWLMTDMFHLNAMALIYKNLEKAVNLKDRDAIDNVGYGQYIAGMGFSNVGLGIVHSMAH S LG A FFDTPHGLANALLLPHVLKFNGKICPDLFRNMGRAMGLDMDNLTDDEAVDKVVDAVRSLAIKIGIPQTLKEIGI K KEDLP MLAH QAIDD VC TAGNPRNVTEQDILALYQEAYE 77 138896515MQNFTFRNPTKLIFGRGQIEQLKEEVPKYGKKVLLVYGGGSIKRNGLYDEVMSLLTDIGA EVVELPGVEPN P RLSTVKKGVDICRREGIEFLLAVGGGS V IDC T KAI AAG AKFDG DPW E FITKKATVTEAL PFGTVLTLAATG S EM N AG SV ITNWET KE KYGW GS PVTFPQFSILDPTYTMTVPKDHTVYGIVDMMSHVFEQYFHHTPNTPLQDRMCEAVLKTVIEAAPKLVDDLENYELRETIMYSGTIALNGFLQMGVRGDWATHDIE H AVS A VYDIPHAGGLAILFPNWMKHVLDENVSRFAQLAVRVFDVDPTGKTERDVALEGIERLRAFWSSLGAPSRLADYGI GEENLELMADKAMAFGEFGRFKTLNRDDVLAILRASL consensusM(T,K)(N,-)(T,-)(QK)(S,T,R)(N,A,I,K)F(F,Y)(I,M)P(P,S)(A,V,S)(N,S,T)(V,L)(F,I)G(R,A,P)G(A,S)V(K,N)EVG(T,A)RL(K,A)(Q,G,D,A)(I,L)G(A,V)(K,T)KAL(I,I)VTDA(F,G)LH(G,S)(T,I)GLSE(E,K)(V,I)(A,S)(K,S,G)(N,I)IR(E,A)AG(V,L) (D,E)(V,A)(A,V,S,L)IFPKA(Q, E)P(D,N) P(A,T)D(T,K)(Q,N)V(H,A)EG(V,L)(D,E)(V,A,I)(F,Y)(K,N)(Q,A)E (N,K) CD(S,A)(L,T)V(S,T)(I,L)GGGSSHD(T,A) (G,A) K(A,G)I (G,A)LV AANGG(R,T,K)I(N,H,Y) DY(Q, E) GV(N,D)(S,V,K)(V,)(E,K)(K,E)(P,Q)(M,V)VP(L,V,Q)(I,V)AI(N,T)TTAGTG S E(T,L) T (S,K,R)(F,L) (A,T)(V,I)ITD(S,T)(A,E)R KV KM(P,A)(V,I) (I,V)D (E,K)(H,K)(I,V)TPT(V,L)(A,S)I(V,N)DPELMV(K,G)(K,M)P(P,A)(G,S)LT(I,A)ATG(M,L)DAL(S,T)HAIEAYV(A,S)(K,T)(G,R,A)ATP(V,I)TDA(F,L)AIQA(M,I)K(L,T)I(N,S)(E,K)YLP(R,K)A(V,F)ANG(E,K)D(I,M)EARE(Q,A,K)MA(Y,F)AQ(Y,S)(M,L)AG(M,V)AFNN(G,A)(G,S)LG(Y,L)VH(S,A)I(S,A)H Q (V,L,F)G G(F,V)Y(K,N)(F,L)(P,Q)HG(I,V)CN(S,A)(V,I)(N,L,V)(M,L)P(H,Y)VC(R,Q,A)FNLI(A,S)(K,R)(T,V)ER(F,Y)A(H,E)IA(E,A)(L,F)LGENV(S,A,D)GLST(A,Y,R)(S,A,E,D)AAE(R,K)(A,T,G)I(K,V,A)A(L,T)(E,Q)R(M,Y,I)(N,A)(K,R)(D,N,S)(F,L)(G,N)IP(S,K,R)G(Y,F)(K,A)(E,A)(M,L)G(V,A) K EEDI(E,V)(L,T)LA(K,N,E) NA(M,Y,F)(Q,N,K,E)D (V,A)(C,T)(T,A)(L,Q)(T,S,D)NPR(V,K)(P,A)(T,K)(V,L)(QE)(D,E)(I,V)(A,I,Q)QIIKNA(M,L).

What is claimed is:
 1. An engineered microbial cell either (a)expressing a non-natural NAD⁺-dependent alcohol dehydrogenase comprisingat least one amino acid substitution as compared to a correspondingalcohol dehydrogenase and capable of greater conversion of methanol orethanol to formaldehyde or acetaldehyde, respectively, as compared to anengineered microbial cell expressing the corresponding alcoholdehydrogenase without amino acid substitution or (b) expressing a firstsequence that is a non-natural NAD⁺-dependent alcohol dehydrogenasecomprising at least one amino acid substitution capable of greaterconversion of methanol or ethanol to formaldehyde or acetaldehyde,respectively, as compared to an engineered microbial cell expressing asecond sequence that is an NAD⁺-dependent alcohol dehydrogenase, whereinthe first and second sequences differ with regards to the at least oneamino acid substitution, wherein the non-natural alcohol dehydrogenasehas a sequence identity of 75% or greater to an NAD⁺-dependent alcoholdehydrogenase template selected from the group consisting of SEQ ID NO:1 (MDH MGA3_17392), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,and SEQ ID NO: 77, or a fragment of said template having saiddehydrogenase activity with an amino-terminal deletion, carboxy-terminaldeletion, or both, the fragment having a sequence identity of 75% orgreater, to the template, and wherein the non-natural alcoholdehydrogenase comprises one or more amino acid substitutions based onformula: R¹XR², wherein R¹ is an original amino acid at position X ofthe template, and R² is the variant amino acid that replaces R¹ at aposition on the template corresponding to X, wherein XR² is selectedfrom the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A,63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T,103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q,117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V,121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R,125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M,129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F,148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M,155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F,163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V,217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V,312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M,355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K,and 379M.
 2. The engineered microbial cell of claim 1 further comprising(c) one or more metabolic pathway transgene(s) encoding a protein of ametabolic pathway that promotes production of a target product orintermediate thereof, (d) a transgene encoding an enzyme to convert theformaldehyde to formate thereby generating reducing equivalents usefulto product the target product and/or able to fix carbon of formate intothe target product, or both (c) and (d).
 3. The engineered microbialcell of claim 1, wherein expression of the non-natural alcoholdehydrogenase provides an increased amount of reducing equivalents foran increase in a target product and/or for increased fixation of carbonfrom the formaldehyde into a target product.
 4. The engineered microbialcell of claim 2 wherein the target product is selected from the groupconsisting of a diol, 1,4-butanediol, 1,3-butanediol, butadiene,succinate, adipate, HMDA, 6-aminocaproic acid (6ACA), methacrylic acid(2-methyl-2-propenoic acid), methacrylate, methyl methacrylate,3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate compoundthereof.
 5. The engineered microbial cell of claim 1 further comprising(c) one or more alcohol metabolic pathway gene(s) encoding a proteinselected from the group consisting of a), a formate dehydrogenase (EM8),a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase(EM11), a S-(hydroxymethyl)glutathione synthase (EM12), aglutathione-dependent formaldehyde dehydrogenase (EM13), aS-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15),and a hydrogenase (EM16); (d) one or more alcohol metabolic pathwaygene(s) encoding a protein selected from the group consisting of asuccinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate(4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), aphosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase(aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); asuccinate reductase (EB9), a succinyl-CoA reductase (alcohol forming)(EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoAsynthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoAreductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1),and a succinyl-CoA synthetase (EB2A), or both (c) and (d), or (e) aformaldehyde assimilation pathway enzyme (FAPE) comprising ahexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase(EF2), a dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4). 6.A composition comprising the engineered microbial cell of claim 1, acell culture composition, optionally comprising a target product orintermediate thereof, or a cell extract thereof.
 7. A method forincreasing the conversion of methanol or ethanol to formaldehyde oracetaldehyde, respectively, comprising a step of (a) culturing theengineered microbial cell of claim 1 in a culture medium comprisingmethanol or ethanol, where in said culturing the cell provides greaterconversion of the methanol or ethanol to formaldehyde or acetaldehyderespectively, as compared to an engineered microbial cell expressing thecorresponding alcohol dehydrogenase without amino acid substitution. 8.The engineered microbial cell of claim 1 wherein the non-natural alcoholdehydrogenase which is capable of at least two fold greater, of at leastthree fold, of at least four fold, of at least five fold, of at leastsix fold, of at least seven fold, at least 8 fold, at least 9 fold, atleast 10 fold, or at least 11 fold, or in the range of two fold totwelve fold greater, in the range of two fold to eleven fold greater, inthe range of two fold to ten fold greater, in the range of two fold tonine fold greater, in the range of two fold to eight fold greater, inthe range of two fold to seven fold greater, in the range of two fold tosix fold greater, in the range of two fold to five fold greater, or inthe range of two fold to four fold greater conversion of methanol orethanol to formaldehyde or acetaldehyde, respectively, in vivo or invitro, as compared to the corresponding alcohol dehydrogenase withoutamino acid substitution.
 9. The engineered microbial cell of claim 1wherein the NAD⁺-dependent non-natural alcohol dehydrogenase has acatalytic efficiency (k_(cat)/K_(m)) for the conversion of methanol toformaldehyde of 8.6×10⁻⁴ or greater.
 10. A method of producing a targetproduct or its intermediate comprising culturing the engineeredmicrobial cell of claim 1 in a culture medium comprising methanol orethanol to produce the target product (TP) or its intermediate (INT).11. A method of preparing a polymer comprising obtaining a targetproduct produced by the engineered microbial cell of claim 1 andpolymerizing the target product, optionally with one or more othermonomeric compounds, to provide a polymeric product.
 12. The engineeredmicrobial cell of claim 1 wherein the non-natural alcohol dehydrogenasehas a sequence identity of 85% or greater, 90% or greater, 92.5% orgreater, 95% or greater, 96% or greater, 97% or greater, 98% or greater,or 99% or greater to an NAD⁺-dependent alcohol dehydrogenase templateselected from the group consisting of SEQ ID NO: 1 (MDH MGA3_17392), SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO: 77 or afragment of said template having said dehydrogenase activity with anamino-terminal deletion, carboxy-terminal deletion, or both, thefragment having a sequence identity of 85% or greater, 90% or greater,92.5% or greater, 95% or greater, 96% or greater, 97% or greater, 98% orgreater, or 99% or greater to the template.
 13. The engineered microbialcell of claim 1 wherein the non-natural alcohol dehydrogenase has asequence identity of 75% or greater to SEQ ID NO: 1 (MDH MGA3_17392) andwherein R¹XR² is selected from the group consisting of (a) S11T, D38N,H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P71I, P71T, P71V,T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L, G107S,L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q, N117Y,Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S, G121T,G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R, N123Y,S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C, K127R,P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y,A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I, L148T,L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M, A155V,K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S, P161V,I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T, L186M,T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M, G270S,G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L, P336L,S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I, N355K,E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K, and K379Mor (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S, L108V, L108W,V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L, G121M, G121R,G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I, N123L, N123R,N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C, K127R, P128A,P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I, V130Y, A134T,S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N, V158E, V158H,V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y, K345E, N355D,V360G, V360K, V360R, V360S, and C361R.
 14. The engineered microbial cellof claim 13 wherein the non-natural alcohol dehydrogenase comprises two,three, four, five, six, seven, eight, nine, ten, eleven or twelve, aminoacid substitutions selected from the group consisting of: (a) S11T,D38N, H42Q, E48D, N53I, E56K, D60E, V61A, I63F, P65Q, D70N, P711, P71T,P71V, T74S, D81G, K84R, E86K, N87K, I94V, S99P, S99T, A103V, I106L,G107S, L108V, L108W, V109Y, N112K, N112R, R115H, I116F, N117D, N117Q,N117Y, Q120H, Q120R, G121A, G121D, G121E, G121L, G121M, G121R, G121S,G121T, G121V, G121W, G121Y, V122A, V122P, N123D, N123I, N123L, N123R,N123Y, S124I, S124L, S124R, V125C, V125G, V125W, E126G, E126V, K127C,K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I,V130Y, A134T, S143T, T145M, T146N, S147R, L148A, L148F, L148G, L148I,L148T, L148V, L148W, A149L, A149M, A149T, A149V, V150A, V150I, T152M,A155V, K157N, V158E, V158H, V158K, V158W, P161A, P161G, P161Q, P161S,P161V, I163F, I163N, I163Q, I163T, D164G, D164N, E165G, K181R, A184T,L186M, T190A, T190S, I199V, Q217K, L226M, G256C, Q267H, G269S, G270M,G270S, G270Y, T296S, R298H, A300T, I302V, G312V, A316V, I323M, F333L,P336L, S337C, G343D, V344A, V344G, K345E, E350K, K354M, N355D, N355I,N355K, E358G, V360A, V360G, V360K, V360R, V360S, C361N, C361R, Q363K,and K379M or (b) D38N, D60E, P71I, P71V, N87K, S99T, A103V, G107S,L108V, L108W, V109Y, R115H, I116F, N117D, N117Q, G121D, G121E, G121L,G121M, G121R, G121S, G121T, G121V, G121W, G121Y, V122P, N123D, N123I,N123L, N123R, N123Y, S124I, S124L, V125C, V125G, V125W, E126G, K127C,K127R, P128A, P128R, P128S, V129A, V129M, V129P, V129S, V130F, V130I,V130Y, A134T, S143T, T146N, A149L, A149M, A149T, A149V, V150A, K157N,V158E, V158H, V158K, V158W, I163Q, D164N, Q267H, G270M, G270S, G270Y,K345E, N355D, V360G, V360K, V360R, V360S and C361R.
 15. The engineeredmicrobial cell of claim 13 wherein the non-natural alcohol dehydrogenasecomprises a set of amino acid substitutions selected from the groupconsisting of (a) D70N, L148G, P161G, V360A; (b) D70N, L148G, V360A,C361N; (c) D70N, L148V, V150I, P161A, V360G; (d) D70N, L148V, V360G; (e)D70N, P161A, V360A; (f) D70N, P161V, V360G, C361N; (g) D70N, V150I,P161A, V360A; (h) D70N, V150I, P161V, V360G, C361N; (i) E48D, L148V,P161A, V360A; (j) L148G, P161A, V360A, C361N; (k) L148G, P161A, V360G;(1) L148G, P161A, V360G, C361N; (m) L148G, P161G, V360A; (n) L148G,P161G, V360G, C361N; (o) L148G, V360A, C361N; (p) L148G, V360G, C361N;(q) L148I, P161G, V360G; (r) L148I, P161V, V360G; (s) L148T, V150I,V360A; (t) L148T, V360G; (u) L148V, P161A, V360A; (v) L148V, V150I,P161A, V360A; (w) L148V, V150I, P161A, V360A, C361N; (x) L148V, V150I,P161A, V360G; (y) L148V, V150I, P161A, V360G, C361N; (z) L148V, V150I,P161A, V360G, C361N; (aa) L148V, V150I, P161G, V360A; (ab) L148V, V150I,P161V, V360G, C361N; (ac) L148W, P161A, V360A, C361N; (ad) N112K, S147R,P161A, V360A; (ae) P161A, Q217K, V360A, C361N; (af) P161A, V360A, C361N;(ag) P161A, V360G; (ah) P161V, E358G, V360G; (ai) P161V, V360A, C361N;(aj) L148W, P161A, V360A, C361N; (ak) N112K, S147R, P161A, V360A; (al)P161A, Q217K, V360A, C361N; (am) P161A, V360A, C361N; (an) P161A, V360G;(ao) P161V, E358G, V360G; (ap) P161V, V360A, C361N; (aq) P161V, V360G;(ar) P65Q, L148G, V150I, P161A, V360G, C361N; (as) S147R, L148A, V150I,P161A, V360G; (at) S147R, L148F, V150I, P161G, V360G; (au) S147R, L148V,P161G, V360A; (av) P161V, V360G; (aw) P65Q, L148G, V150I, P161A, V360G,C361N; (ax) S147R, L148A, V150I, P161A, V360G; (ay) S147R, L148F, V150I,P161G, V360G; (az) S147R, L148V, P161G, V360A; (aaa) S147R, L148V,P161V, V360G; (aab) S147R, L148V, V150I, P161A, C361N; (aac) S147R,L148V, V150I, P161G, V360G; (aad) S147R, P161A, V360A; (aae) S147R,P161A, V360A, C361N; (aaf) S147R, P161A, V360G; (aag) S147R, P161V,V360G; (aah) S147R, P161V, V360G, C361N; (aai) S147R, V150I, P161V,V360A; (aaj) S147R, V150I, V360A, C361N; (aak) T145M, L148I, V360G;(aal) V150I, I302V, V360G, C361N; (aam) V150I, P161A, C361N; (aan)V150I, P161G, V360A, C361N; (aao) V150I, P161G, V360G; (aap) V150I,P161G, V360G, C361N; (aaq) V150I, P161V, C361N; (aar) V150I, P161V,K354R, V360A, C361N; (aas) V150I, P161V, V360A, C361N; (aat) V150I,P161V, V360G, C361N; (aau) V150I, V360A, C361N; (aav) V150I, V360G;(aaw) S11T, T74S, G269S, V344A; (aax) K84R, I163T; (aay) V122A, I163N;(aaz) G107S, F333L; (aaaa) V129M, T152M, G343D; (aaab) I63F, N355K;(aaac) G107S, F333L; (aaad) E86K, S99T, A149V; (aaae) N53I, V158E;(aaaf) N355I, K379M; (aaag) H42Q, G107S; (aaah) Q120H, I163N; (aaai)A149V, I323M; (aaaj) G107S, F333L; (aaak) D164G, K181R; (aaal) A155V,R298H, N355D; (aaam) N123D, E165G; (aaan) I163F, L186M; (aaao) G121A,T296S; (aaap) I94V, S99P, N123I; (aaaq) E126V, V129M, V344G; (aaar)Q120R, S143T; (aaas) G256C, A316V; (aaat) P161Q, G312V; (aaau) L226M,A300T, V360A; (aaav) S337C, E350K, N355D, Q363K; (aaaw) D81G, V158E;(aaax) I106L, N117Y, E126V; (aaay) G107S, G121D; (aaaz) V61A, V158E;(aaaaa) N53I, V158E; (aaaab) N117Y, T190S; (aaaac) S124R, I199V; (aaaad)K354M, C361R; (aaaae) A184T, C361R; (aaaag) E56K, Q267H; (aaaag) S124R,E126G; (aaaah) T190A, N355K; (aaaai) P71T, F333L; (aaaaj) G107S, F333L;and (aaaak) N123I, P336L, (aaaal) D38D/A149V, (aaaam) D38N/V163V,(aaaan) D73D/L108V, (aaaao) G121R/P161S, and (aaaap) N112R/P161S. 16.The engineered microbial cell of claim 1 wherein the non-natural alcoholdehydrogenase comprises a sequence motif selected from the groupconsisting of (a) TNA and VTNAF (SEQ ID NO: 79); (b) VEV and GVEVA (SEQID NO: 80); (c) DIA, PDIAD (SEQ ID NO: 81), DVA, and PDVAD (SEQ ID NO:82); (d) EKC and QEKCD (SEQ ID NO: 83); (e) STH and GSTHD (SEQ ID NO:84); (f) TVK and DTVKA (SEQ ID NO: 85); (g) SLV, GVV, GWV, GLY, ISLVA(SEQ ID NO: 86), IGVVA (SEQ ID NO: 87), IGWVA (SEQ ID NO: 88), and IGLYA(SEQ ID NO: 89); (h) HIN, RFN, RID, RIQ, GHIND (SEQ ID NO: 90), GRFND(SEQ ID NO: 91), GRIDD (SEQ ID NO: 92), and GRIQD (SEQ ID NO: 93); (i)DVNSVEKPVV (SEQ ID NO: 94), EVNSVEKPVV (SEQ ID NO: 95), LVNSVEKPVV (SEQID NO: 96), MVNSVEKPVV (SEQ ID NO: 97), RVNSVEKPVV (SEQ ID NO: 98),SVNSVEKPVV (SEQ ID NO: 99), TVNSVEKPVV (SEQ ID NO: 100), VVNSVEKPVV (SEQID NO: 101), WVNSVEKPVV (SEQ ID NO: 102), YVNSVEKPVV (SEQ ID NO: 103),GPNSVEKPVV (SEQ ID NO: 104), GVDSVEKPVV (SEQ ID NO: 105), GVISVEKPVV(SEQ ID NO: 106), GVLSVEKPVV (SEQ ID NO: 107), GVRSVEKPVV (SEQ ID NO:108), GVYSVEKPVV (SEQ ID NO: 109), GVNIVEKPVV (SEQ ID NO: 110),GVNLVEKPVV (SEQ ID NO: 111), GVNSCEKPVV (SEQ ID NO: 112), GVNSGEKPVV(SEQ ID NO: 113), GVNSWEKPVV (SEQ ID NO: 114), GVNSVGKPVV (SEQ ID NO:115), GVNSVECPVV (SEQ ID NO: 116), GVNSVERPVV (SEQ ID NO: 117),GVNSVEKAVV (SEQ ID NO: 118), GVNSVEKRVV (SEQ ID NO: 119), GVNSVEKSVV(SEQ ID NO: 120), GVNSVEKPAV (SEQ ID NO: 121), GVNSVEKPMV (SEQ ID NO:122), GVNSVEKPPV (SEQ ID NO: 123), GVNSVEKPSV (SEQ ID NO: 124),GVNSVEKPVF (SEQ ID NO: 125), GVNSVEKPVI (SEQ ID NO: 126), and GVNSVEKPVY(SEQ ID NO: 127); (j) TETT (SEQ ID NO: 128), SETN (SEQ ID NO: 129),GTETTS (SEQ ID NO: 130), and GSETNS (SEQ ID NO: 131); (k) LLVI (SEQ IDNO: 132), LMVI (SEQ ID NO: 133), LTVI (SEQ ID NO: 134), LVVI (SEQ ID NO:135), and LAAI (SEQ ID NO: 136); (1) NVKMPVID (SEQ ID NO: 137), KEKMPVID(SEQ ID NO: 138), KHKMPVID (SEQ ID NO: 139), KKKMPVID (SEQ ID NO: 140),KWKMPVID (SEQ ID NO: 141), KVKMPVQD (SEQ ID NO: 142), and KVKMPVIN (SEQID NO: 143); (m) HVGG (SEQ ID NO: 144), QVGM (SEQ ID NO: 145), QVGS (SEQID NO: 146), and QVGY (SEQ ID NO: 147); (n) VEE and GVEEE (SEQ ID NO:148); or (o) DAYEDVC (SEQ ID NO: 149), NAYEDGC (SEQ ID NO: 150), NAYEDKC(SEQ ID NO: 151), and NAYEDRC (SEQ ID NO: 152), and NAYEDSC (SEQ ID NO:153), and NAYEDVR (SEQ ID NO: 154).
 17. The engineered microbial cell ofclaim 1 selected from the genus Acinetobacter, Actinobacillus,Escherichia, Bacillus, Clostridium and Streptomyces.
 18. The engineeredmicrobial cell of claim 12 wherein the non-natural alcohol dehydrogenasehas a sequence identity of 90% or greater, 92.5% or greater, 95% orgreater, 96% or greater, 97% or greater, 98% or greater, or 99% orgreater to an NAD⁺-dependent alcohol dehydrogenase template selectedfrom the group consisting of SEQ ID NO: 1 (MDH MGA3_17392), SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO:
 77. 19. Theengineered microbial cell of claim 1 wherein the non-natural alcoholdehydrogenase has a sequence identity of 75% or greater to SEQ ID NO: 1(MDH MGA3_17392).